Immunotherapy using interleukin 13 receptor subunit alpha 2

ABSTRACT

A method for stimulating a immune response against IL-13Rα2 in a subject having or at risk for developing a disease having cells expressing IL-13Rα2 includes the steps of formulating the anti-cancer vaccine outside of the subject and administering the vaccine to the subject in an amount sufficient to stimulate an immune response against IL-13Rα2 in the subject. A composition for stimulating a immune response against IL-13Rα2 in a subject having or at risk for developing a disease having cells expressing IL-13Rα2 includes an isolated agent that can stimulate immune response against IL-13α2.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. provisionalapplication serial No. 60/181,000 filed Feb. 8, 2000.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under grantnumber CA74154 awarded by the National Cancer Institute of the NationalInstitutes of Health. The Government may have certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the fields of biology,immunology, medicine, and oncology. More particularly, the inventionrelates to the use of the interleukin 13 (IL-13) receptor subunit alpha2 (IL-13Rα2) as an immune system modulator and target for vaccines forthe treatment and prevention of cancer.

BACKGROUND

[0004] Cancer is presently the second leading cause of death indeveloped nations. Wingo et al., J. Reg. Management, 25:43-51 (1998).Despite recent research that has revealed many of the molecularmechanisms of tumorigenesis, few new treatments have achieved widespreadclinical success in treating solid tumors. The mainstay treatments formost malignancies thus remain gross resection, chemotherapy, andradiotherapy. While increasingly successful, each of these treatmentsstill causes numerous undesired side effects. The primary cause of thisis that none of these conventional methods specifically targets onlydiseased cells. For example, surgery results in pain, traumatic injuryto healthy tissue, and scarring. Radiotherapy and chemotherapy causenausea, immune suppression, gastric ulceration and secondarytumorigenesis.

[0005] In an effort to develop techniques to more specifically targetdiseased cells, progress in tumor immunology has led to the discovery ofantigens that are preferentially or specifically expressed on cancercells. These tumor-associated antigens (TAA) or tumor-specific antigens(TSA) have been used as antigenic agents in cancer vaccines designed tostimulate an immune response selectively directed against cancer cellsexpressing such antigens. See, Tumor Immunology: Immunotherapy andCancer Vaccines, A. G. Dalgleish and M. J. Browning, eds., CambridgeUniversity Press, 1996; Immunotherapy in Cancer, M. Gore and P. Riches,eds., John Wiley & Son Ltd., 1996; Maeurer et al., Melanoma Res.,6:11-24 (1996). Among the most widely studied of these antigens aremelanoma associated antigens, prostate specific antigen (PSA), E6 andE7, carcinoembryonic antigen (CEA), p53, and gangliosides (e.g., GM2).More recent studies have shown that certain TAAs and TSAs areparticularly effective at stimulating specific immune responses.

[0006] For example, pioneering research with melanoma associatedantigens led to the identification of MAGE-1 (Melanoma Antigen 1) as aT-cell activating TSA. Traversari et al., Immunogenetics, 35: 145-152,1992. Subsequently other groups using similar techniques identifiedother T-cell activating melanoma antigens including other MAGEs, MART-1,glycoprotein 100 (gp100), tyrosinase, BAGE, and GAGE. Reviewed byMaeurer et al., supra. One of the most exciting recent findings incancer immunology came after the SEREX (for serological analysis ofrecombinant cDNA expression libraries) technique was developed. Sahin etal., Proc. Natl. Acad. Sci. USA, 92: 11810-11813, 1995. The SEREXtechnique involves screening a cDNA expression library of an autologoustumor by exposing the library to antibodies contained in a patient'ssera. Several active cancer antigens have been identified using thistechnique. See, Old, L. J. and T. C. Chen, J. Exp. Med., 187: 1163-1167,1998. Moreover, SEREX analysis showed that patients produce a high titerof IgG antibodies against cancer antigens- a finding that indicated thathelper T cells (e.g., CD4+ T cells) and B cells cooperate in stimulatingan immune response against the cancer.

[0007] In addition, SEREX analyses led to the identification of a groupof cancer antigens termed “cancer/testis” antigens (CTAs). CTAs shareseveral common features including (a) among normal organs, almostexclusive expression in the testis, (b) expression in a wide variety oftumors, (c) presence of multiple members in each identified family, and(d) localization of their genes to the X chromosome (with the notableexception of SCP 1). Chen et al., J. Biol. Chem., 273: 17618-17625,1998. Based on the foregoing criteria, several previously identifiedTAAs or TSAs (e.g., MAGE, BAGE and GAGE) were re-discovered as CTAs.Notably, unlike many non-CTA antigens, most of these previouslyidentified CTAs as well as newly identified CTAs (e.g., SSX2, NY-ESO-1,SCP1 and CT7) have unequivocally been shown to stimulate an immuneresponse in a subject.

SUMMARY

[0008] The invention relates to the discovery that IL-13Rα2 is acancer/testis antigen. This discovery is important because, in contrastto most other cancer-associated agents, most of the cancer/testisantigens so far tested as active immunotherapy agents against cancerhave proven very effective in stimulating anti-cancer immune responsesin subjects. Thus, the present discovery provides methods andcompositions for preventing and/or treating cancers that expressIL-13Rα2.

[0009] In particular, the invention relates to the treatment and/orprevention of high-grade gliomas (HGG) in a subject as HGG cells havebeen shown to express high levels of IL-13Rα2 on their surfaces. HumanHGG are rapidly progressing heterogeneous brain tumors of astroglialorigin. The present invention is especially important because noeffective modalities for treating HGG are yet accepted for clinical use.Previously, it was shown that the vast majority of HGG patientsover-express a more restrictive receptor for IL-13, that is a receptorthat binds IL-13 in an IL-4 independent manner. Recently, a new IL-13binding protein, termed IL-13Rα2, was cloned. This protein was shown tohave affinity for IL-13 but not IL-4. In a rough comparison, thischaracteristic relates to the more restrictive receptor for IL-13expressed on HGG. Here we demonstrate that, IL-13Rα2 serves as aselective target for HGG and other cancers that express IL-13Rα2because, as described in more detail below, with the exception oftestis, normal human tissue expresses little or no IL-13Rα2. Andalthough many normal tissues express a receptor that binds IL-13, thisreceptor (sometimes termed the “shared” receptor because it binds bothIL-13 and IL-4) differs functionally from IL-13Rα2 (believed to be the“restrictive” receptor) in that the shared receptor binds both IL-13 andIL-4, while the restrictive receptor binds only IL-13. The two receptorsalso differ structurally, with the restrictive receptor being a 42 kDamonomer and the shared receptor being a heterodimer composed of a 45 kDacomponent (termed IL-13Rα1) and a 140 kDa component (termed IL-4Rα).

[0010] As indicated above, our tissue distributions studies showed that,among normal tissues, IL-13Rα2 is strongly expressed only in testis.This finding along with the showing that (a) IL-13Rα2 is preferentiallyover-expressed on HGG but not normal central nervous system (CNS) tissueand (b) that the IL-13Rα2 gene is localized to chromosome X, indicatesthat IL-13Rα2 is a CTA. Because other CTAs have proven to stimulate astrong immune response against cancer cells, the present inventionprovides methods and compositions useful for generating or increasing ananti-cancer immune response in a subject.

[0011] Accordingly, in one aspect the invention features a method forstimulating a immune response against IL-13Rα2 in a subject having or atrisk for developing a disease having cells expressing IL-13Rα2. Themethod includes the steps of: (a) formulating an anti-cancer vaccineoutside of the subject, the vaccine including an agent that canstimulate an immune response against IL-13Rα2 when administered to ananimal; and (b) administering the vaccine to the subject in an amountsufficient to stimulate an immune response against IL-13Rα2 in thesubject.

[0012] In another aspect the invention features a composition forstimulating an immune response against IL-13Rα2 when administered to ananimal. The composition includes: (a) an isolated agent that canstimulate an immune response against IL-13Rα2 when administered to ananimal; and (b) a pharmaceutically acceptable carrier.

[0013] In both of the foregoing method and composition, the agent thatcan stimulate an id immune response against IL-13Rα2 can include apeptide including at least seven contiguous amino acids of SEQ ID NO: 1.For example, the agent can be a protein including the amino acidsequence of SEQ ID NO: 1. The agent can also take the form of a nucleicacid that encodes a peptide including at least seven contiguous aminoacids of SEQ ID NO: 1. Such a nucleic acid can be used as a naked DNA orin an expression vector construct including the nucleic acid. The agentthat can stimulate an immune response against IL-13Rα2 can also be acell. This cell can be one that expresses a peptide including at leastseven contiguous amino acids of SEQ ID NO: 1, or one into which apurified nucleic acid that encodes a peptide including at least sevencontiguous amino acids of SEQ ID NO: 1 has been introduced.

[0014] The vaccines and compositions within the invention can furtherinclude an adjuvant such as an aluminum salt; an oil-in-water emulsion;a composition including saponin; a composition including a bacterialprotein; or a cytokine.

[0015] The method of the invention can further include a step ofproviding a subject (e.g., a human being) having or at risk fordeveloping a cancer having cells expressing IL-13Rα2 (e.g., gliomacells). Also in the method, the step of administering the vaccine to thesubject in an amount sufficient to stimulate an immune response againstIL-13Rα2 in the subject can include administering the vaccine in atleast a first dose and a second dose, wherein the first dose isadministered to the subject at least 24 hours before the second dose isadministered to the subject.

[0016] Unless otherwise defined, all technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Definitions of molecular biologyterms can be found, for example, in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994. Standardone-letter nomenclature for nucleotide bases, and one- and three-letternomenclature for amino acid residues are used.

[0017] As used herein, a “nucleic acid” means a chain of two or morenucleotides. For example, RNA (ribonucleic acid) and DNA(deoxyribonucleic acid) are nucleic acids. An “isolated” nucleic acid isone that has been substantially separated or purified away from othernucleic acid sequences in the cell of the organism in which the nucleicacid naturally occurs, i.e., other chromosomal and extrachromosomal DNAand RNA, e.g., by conventional nucleic acid purification methods. Theterm therefore includes a recombinant nucleic acid molecule incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote. It includes aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), or a restriction fragment.It also includes recombinant nucleic acid molecules and chemicallysynthesized nucleic acid molecules. A “recombinant” nucleic acidmolecule is one made by an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

[0018] When referring to a nucleic acid molecule or polypeptide, theterm “native” refers to a naturally-occurring (e.g., a “wild-type”)nucleic acid or polypeptide. A “homolog” of an IL-13Rα2 gene is a genesequence encoding an IL-13Rα2 polypeptide isolated from a species otherthan Homo sapiens. By the phrase “naked nucleic acid” is meant anisolated nucleic acid not incorporated in an expression vector.

[0019] By the terms “IL-13Rα2 gene” or “IL-13Rα2 polynucleotide” ismeant a native IL-13Rα2 encoding nucleic acid sequence (e.g., theIL-13Rα2 cDNA sequence shown as SEQ ID NO: 2 (FIG. 2)), genomicsequences from which IL-13Rα2 cDNA can be transcribed, and/or allelicvariants and homologs of the foregoing.

[0020] As used herein, “protein,” “peptide,” or “polypeptide” means anypeptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.Generally, the term “peptide” is used herein to refer to amino acidchains less than about 25 amino acid residues in length, while the terms“protein” and “polypeptide” are used to refer to larger amino acidchains. When referring to a protein or peptide, the term “isolated”means proteins or peptides that are isolated from other cellularproteins or are made synthetically. The term thus encompasses bothpurified and recombinant polypeptides. The term “recombinant protein” or“recombinant peptide” refers to a protein or peptide that is produced byrecombinant nucleic acid techniques, wherein generally, a nucleic acidencoding the peptide or protein is inserted into a suitable expressionvector which is in turn used to transform a host cell such that, whencultured under appropriate conditions, the cell produces the peptide orprotein.

[0021] By “IL-13Rα2 protein” “IL-13Rα2 polypeptide,” or simply“IL-13Rα2” is meant an expression product of an IL-13Rα2 gene such asthe protein of SEQ ID NO: 1 (FIG. 1); or a ffi protein that shares atleast 65% (but preferably 75, 80, 85, 90, 95, 96, 97 ,98, or 99%) aminoacid sequence identity with SEQ ID NO: 1 and cross-reacts withantibodies that specifically bind the protein of SEQ ID NO: 1.

[0022] As used herein, “sequence identity” means the percentage ofidentical subunits at corresponding positions in two sequences when thetwo sequences are aligned to maximize subunit matching, i.e., takinginto account gaps and insertions. When a subunit position in both of thetwo sequences is occupied by the same monomeric subunit, e.g., if agiven position is occupied by an adenine in each of two DNA molecules,then the molecules are identical at that position. For example, if 7positions in a sequence 10 nucleotides in length are identical to thecorresponding positions in a second 10-nucleotide sequence, then the twosequences have 70% sequence identity. Preferably, the length of thecompared sequences is at least 60 nucleotides, more preferably at least75 nucleotides, and most preferably 100 nucleotides. Sequence identityis typically measured using sequence analysis software (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705).

[0023] A first nucleic-acid sequence is “operably” linked with a secondnucleic-acid sequence when the first nucleic-acid sequence is placed ina functional relationship with the second nucleic-acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably linked DNA sequences are contiguous and, wherenecessary to join two protein coding regions, in reading frame.

[0024] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. A vector capable of directing the expression of a gene towhich it is operatively linked is referred to herein as an “expressionvector.” As used herein, the term “promoter” means a nucleic acidsequence that regulates expression of a selected nucleic acid sequenceoperably linked to the promoter, and which effects expression of theselected nucleic acid sequence in cells. The term encompasses “tissuespecific” promoters, i.e. promoters, which effect expression of theselected nucleic acid sequence only in specific cells (e.g. cells of aspecific tissue). The term also covers so-called “leaky” promoters,which regulate expression of a selected nucleic acid primarily in onetissue, but cause expression in other tissues as well. The term alsoencompasses both non-tissue specific promoters and promoters that areconstitutively active and inducible.

[0025] By the phrase “stimulating an immune response” is meant elicitingor increasing the activation of a lymphocyte (e.g., a B cell or T cell)or other immune system component. The stimulation of an immune responseagainst a specific antigen can be measured as an increase in antibodytiter against that antigen or the activation of one or more lymphocyteshaving a surface receptor specific for the antigen. Activation oflymphocytes can be determined by conventional assays, e.g., theinduction of mitosis, secretion of cytokines, modulation of cell surfacemolecule expression, secretion of immunoglobulin (B cells), andincreased killing of target cells (cytotoxic T cells).

[0026] As used herein, “bind,” “binds,” or “interacts with” means thatone molecule recognizes and adheres to a particular second molecule in asample, but does not substantially recognize or adhere to otherstructurally unrelated molecules in the sample. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁵ to 10⁶ liters/mole for that secondmolecule.

[0027] By the term “antibody” is meant any antigen-binding peptidederived from an immunoglobulin. The term includes polyclonal antisera,monoclonal antibodies, fragments of immunoglobulins produced byenzymatic digestion (e.g., Fab fragments) or genetic engineering (e.g.,sFv fragments).

[0028] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The invention is pointed out with particularity in the appendedclaims. The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

[0030]FIG. 1 is the amino acid sequence of the native H. sapiensIL-13Rα2 protein.

[0031]FIG. 2 is the nucleic acid sequence of a cDNA corresponding to anative mRNA encoding the native H. sapiens IL-13Rα2 protein.

[0032]FIG. 3 is a schematic representation of two types of IL13receptors: the shared with IL4 physiological, heterodimeric IL13/4R, andan IL4-independent monomeric, HGG-associated IL13R. A, 140-kDa IL4Rα-chain. B, 45-kDa IL13R α1-chain; A and B constitute the elements ofthe heterodimeric high affinity IL13/4R. C, a 42-kDa monomer of IL13Rα2.

[0033]FIG. 4 is a Northern blot analysis of human IL13Rα2 transcripts(closed figure) in series of CNS (panels I and II) and peripheraltissues (panels III and IV). The migration position of MRNA is shown inkilobases. Films were exposed for 2 weeks.

[0034]FIG. 5 is a Northern blot analysis of human IL13Rα2 transcripts(closed figure) in series of CNS (panels I and II) and peripheraltissues (panels III and IV). The migration position of mRNA is shown inkilobases. Films were exposed for 2 weeks except for membranes shown inpanels III and IV, which were exposed for 3 days.

[0035]FIG. 6 is a Northern blot analysis of human 140-kDa IL4R α-chaintranscripts (closed figure) in series of CNS (panels I and II) andperipheral tissues (panel IV). The migration position of mRNA is shownin kilobases. Films were exposed for 2 weeks.

[0036]FIG. 7 is a Northern blot analysis of human P-actin transcripts inCNS (panels I and II) and peripheral tissues (panel IV). The migrationposition of rnRNA is shown in kilobases. Films were exposed for 1-3hours.

[0037]FIG. 8 is a Northern blot analysis of transcripts of different IL13 receptors in malignant glioma cells (G-48, A-172 MG, U-373 MG, andU-251 MG), normal human umbilical vein endothelial cells (HUVEC) and insurgical specimens of GBM and normal human brain. The migration positionof mRNA is shown in kilobases. Films were exposed for 2 weeks, exceptfor actin (1 hr).

DETAILED DESCRIPTION

[0038] The invention encompasses compositions and methods relating tostimulating an immune response against IL-13Rα2 in a subject having orbeing at risk for developing a cancer or other disease having cellsexpressing IL-13Rα2. The below described preferred LO embodimentsillustrate adaptations of these compositions and methods. Nonetheless,from the description of these embodiments, other aspects of theinvention can be made and/or practiced based on the description providedbelow.

[0039] Biological Methods

[0040] Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook el al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981,andMatteuccietal., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis ofnucleic acids can be performed, for example, on commercial automatedoligonucleotide synthesizers. Immunological methods (e.g., preparationof antigen-specific antibodies, immunoprecipitation, and immunoblotting)are described, e.g., in Current Protocols in Immunology, ed. Coligan etal., John Wiley & Sons, New York, 1991; and Methods of ImmunologicalAnalysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.Conventional methods of gene transfer and gene therapy can also beadapted for use in the present invention. See, e.g., Gene Therapy:Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999;Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D.Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy,ed. C. P. Hodgson, Springer Verlag, 1996.

[0041] Identification of IL-13Rα2 as a Cancer/Testis Antigen

[0042] As its name implies, IL-13Rα2 is a receptor for the lymphokineIL-13. IL-13 has been identified as a homologue of IL-4 that is secretedby both B and T cells. Minty et al., Nature, 36: 248-251, 1993; McKenzieet al., Proc. Natl. Acad. Sci. USA, 90: 3735-3739, 1993. Several typesof normal cells contain an IL-13 receptor termed the shared IL-13/IL-4receptor, which is a heterodimer that includes an IL-13 bindingsubcomponent named IL-13Rα1 (Interleukin 13 receptor alpha one). Hiltonet al., Proc. Natl. Acad. Sci. USA, 93: 497-501, 1996; Aman et al., J.Biol. Chem., 271: 29265-29270, 1996; Miloux et al., FEBS Letters, 40:163-166, 1997. In addition to IL-13Rα1, the shared receptor alsoincludes a - protein referred to as p140 (or IL-4Ra), the subcomponentresponsible for IL-4 binding. Idzerda et al., J. Exp. Med., 171:861-873, 1990; Hilton et al., Proc. Natl. Acad. Sci. USA, 93: 497-501,1996; Debinski et al., Nature Biotech., 16: 449-453, 1995; Zurawski etal., EMBO J., 12: 2663-2670, 1993; Minty et al., Nature, 36: 248-251,1993. Exposing cells to IL-13 results in responses very similar to thoseresponses that occur after exposure to IL-4. Zurawski, G., and J.E. deVries, Stem Cells. 12: 169-174, 1994. Examples of cellular responsesresulting from both IL-13 and IL-4 exposure include enhanced expressionof CD72, IgM, and MHC class II antigen, as well as induced CD23expression and IgE heavy-chain gene production in B lymphocytes. Id.

[0043] In an interesting development, it was found that IL-13Rα1 was notthe only IL-13 binding site that existed on cells. In previous studies,it was demonstrated that many cancers, most notably HGG, are capable ofbinding IL-13. Debinski et al., Clin. Cancer Res., 1:1253-1258, 1995;Debinski et al., J. Biol. Chem., 271: 22428-22433, 1996; Debinski etal., Nature Biotech., 16: 449-453, 1998; Debinski et al., Critic Rev.Oncogen., 9: 256-268, 1998; Debinski et al., Clin. Cancer Res., 5:985-990, 1999. Through these studies, it became increasingly clear thatthe IL-13 binding capacity of many of these tumors was not mediatedthrough the shared IL-13/IL-4 receptor (i.e., the receptor now known tobe a heterodimer composed of IL-13Rα1/p140). Notably, in lymphoid cellsthat contain the shared receptor, saturating the receptors with IL-4blocked IL-13 binding. Zurawski et al., EMBO J., 12: 2663-2670, 1993.This was not the case using HGG cells, where IL-13 binding was unalteredeven where a large excess of IL-4 used in neutralization assays.Debinski et al., Clin. Research Res., 1: 1253-1258, 1995; Debinski etal., J. Biol. Chem., 271: 22428-22433, 1996; Debinski et al., NatureBiotech., 16: 449-453, 1998. In further experiments, rationally designedIL-13 mutants were generated that maintained their ability to bindglioblastoma (HGG) cells but lost their ability to interact and causesignaling in cells expressing only the IL-4/IL-13 shared receptor.Debinski et al., Nature Biotech., 16: 449-453, 1998; Thompson, J. P. andW. Debinski, J. Biol. Chem., 274: 29944-29950, 1999; Debinski, W., andJ. P. Thompson, Clin. Cancer Res., 5: 3143s-3147s, 1999. This evidencesupported the existence of an additional IL-13 binding protein,unrelated to known IL-4 binding proteins. Additional evidence wasderived when a novel IL-13 binding protein on cells of renal cellcarcinoma metastases (Caki-1 cells) was isolated and the gene encodingthe protein cloned. Caput et al., J. Biol. Chem., 271:16921, 1996. Thegene encoding this protein, termed IL-13Rα2, was subsequently cloned andsequenced. Id. This novel IL-13 binding protein, referred to herein asIL-13Rα2, was shown not to specifically bind IL-4. The proposedstructures of the shared -15- IL-13/4 receptor and the IL-4-independentreceptor for IL-13 are shown in FIG. 3.

[0044] To investigate whether this newly discovered receptor is presentin HGG, we evaluated its gene expression in HGG established cell lines,and HGG explant cells and tumor specimens. In addition to these studieson HGG, we screened a plethora of normal central nervous system (CNS)tissues and peripheral organs for the MRNA transcripts of IL-13Rα2 inorder to characterize the normal tissue expression pattern of this newreceptor in detail. From these studies, we discovered that IL-13Rα2expression is virtually absent in all normal adult tissue except testis.In earlier studies, the gene encoding IL-13Rα2 was localized to the Xchromosome. Guo et al., Genomics, 42: 141-145, 1997.

[0045] Accordingly, our discovery allowed us to characterize theIL-13Rα2 protein as a member of the CTA group of tumor antigens.Moreover, because IL-13Rα2 is a transmembrane receptor, it is exposed tothe extracellular environment independently of MHC presentation. Thus,in contrast to intracellular antigens that must be displayed as apeptide fragment in complex with an MHC molecule on the cell surface tobe recognized by immune system components, cytotoxic agents orantibodies can be directly targeted to cancer cells bearing IL-13Rα2 ontheir surface. This discovery that IL-13Rα2 is a CTA associated with HGGis significant because no other HGG-associated antigens of thisprevalence are known that could serve as a basis for a rational designof anti-glioma vaccines.

[0046] Vaccines

[0047] The invention provides vaccines that can stimulate an immuneresponse against IL-13Rα2 in a subject when administered to the subject.Vaccines within the invention include an antigenic agent which can takethe form of any substance that can evoke or increase an immune responseagainst IL-13Rα2 when introduced into a subject. Typical immuneresponses include (a) the production of, or increase in titer of,antibodies that specifically bind IL-13Rα2 and (b) the activation of Tlymphocytes (e.g., to kill a target cell or provide help in theactivation of antibody production in B lymphocytes). A number ofdifferent antigenic agents have been shown to be effective instimulating an immune response against a protein antigen, including, forexample, protein- and peptide-based vaccines, tumor-cell vaccines,dendritic cell/gene therapy vaccines and DNA/viral vaccines. See, e.g.,Greten, T. F. and E. M. Jaffee, J. Clin. Oncol., 17: 1047-1060, 1999. Inaddition to the foregoing, various substances such as adjuvants andexcipients/carriers can be included in the vaccine compositions of theinvention to non-specifically enhance the antigen-specific immuneresponse stimulated by the antigenic agent and to facilitate delivery ofthe other components of the vaccine to a subject.

[0048] Protein/Peptide Based Vaccines

[0049] The antigenic agent for use in the vaccines of the invention cantake the form of the native IL-13Rα2 (SEQ ID NO:1) or a peptide fragmentof IL-13Rα2. Vaccines made with the whole protein antigen areadvantageous because they have the capability of stimulating an immuneresponse against all of the potential antigenic sites expressed by theprotein. Vaccines made with peptide antigens (e.g., 7-15 or 8-12contiguous amino acids of the whole protein), on the other hand, willgenerally stimulate an immune response against fewer than all of thepotential antigenic sites expressed by the protein. Peptide-basedvaccines are sometimes advantageous over whole protein-based vaccineswhere it is desired to more specifically target the stimulated immuneresponse, e.g., to avoid undesired cross reactions. For example,peptides for use in the vaccine can be selected to correspond to (1)specific epitopes of the antigens that are known to be presented by MHCclass I or MHC class II molecules, or (2) a modified form of an epitopethat either exhibits an increased stability in vivo or a higher bindingaffinity for an MHC molecule than the native epitope, while still beingcapable of specific activation of T-cells. See, Ayyoub et al., J. Biol.Chem., 274: 10227-10234, 1999; Parkhurst et al., Immunol., 157:2539-2548, 1996. Peptide-based vaccines have been shown to circumventimmune tolerance to the intact proteins. Disis et al., J. Immunol., 156:3151-3158, 1996. In addition to vaccines composed of only one type ofpeptide fragment, other vaccines within the invention also include thosemade up of a cocktail of several different peptides derived fromIL-13Rα2.

[0050] As indicated above, vaccines with in the invention can include anIL-13Rα2 protein as an antigenic agent. Preferred forms of IL-13Rα2protein include a purified native IL-13Rα2 protein that has the aminoacid sequence shown in FIG. 1 (SEQ ID NO: 1). Variants of the nativeIL-13Rα2 protein such as fragments, analogs and derivatives of nativeIL-13Rα2 are also contemplated for use as an antigenic agent in thevaccines of the invention. Such variants include, e.g., a polypeptideencoded by a naturally occurring allelic variant of the native IL-13Rα2gene, a polypeptide encoded by a homolog of the native IL-13Rα2 gene,and a polypeptide encoded by a non-naturally occurring variant of thenative IL-13Rα2 gene. Preferred versions of such variants are those thatare able to stimulate an immune response to native IL-13Rα2 uponadministration to a subject as part of a vaccine.

[0051] IL-13Rα2 protein variants have a peptide sequence that differsfrom the native IL-13Rα2 protein in one or more amino acids. The peptidesequence of such variants can feature a deletion, addition, orsubstitution of one or more amino acids of the native IL-13Rα2polypeptide. Amino acid insertions are preferably of about 1 to 4contiguous amino acids, and deletions are preferably of about 1 to 10contiguous amino acids. In some applications, variant IL-13Rα2 proteinssubstantially maintain a native IL-13Rα2 protein functional activity(e.g., the ability to specifically bind IL-13). For other applications,variant IL-13Rα2 proteins lack or feature a significant reduction in anIL-13Rα2 protein functional activity. Where it is desired to retain afunctional activity of native IL-13Rα2 protein, preferred IL-13Rα2protein variants can be made by expressing nucleic acid molecules withinthe invention that feature silent or conservative changes. VariantIL-13Rα2 proteins with substantial changes in functional activity can bemade by expressing nucleic acid molecules within the invention thatfeature less than conservative changes.

[0052] IL-13Rα2 protein fragments corresponding to one or moreparticular motifs (e.g., those likely to bind with high affinity to MHCmolecules) and/or domains are within the invention as are those ofarbitrary sizes. For example, peptide fragments of IL-13Rα2 proteinconsisting of at least 5, 10, 25, 30, 40, 50, 50, 70, 75, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 300 or more contiguous amino acids of the IL-13Rα2protein are within the scope of the present invention. Fragments ofbetween 7 and 15 amino acids (preferably 8-12 amino acids) in length(e.g., those sized to fit in the grooves of MHC molecules) are preferredas peptides of such size have been shown to serve as efficientimmunogenic agents. Methods for identifying efficiently immunogenicpeptides of a whole protein are known in the art, e.g., usingamphipathicity algorithms. See, e.g., Berzofsky, J. A., Ann. N. Y. Acad.Sci., 12:256, 1993; U.S. Pat. Nos. 5,976,541 and 5,980,899. Peptidesthat are most immunogenic in a subject can also be determined bypreparing a series of overlapping peptide fragments (e.g., 7-30 aminocontiguous amino acids long) of the whole antigen, administering thesubject (or a series of genetically similar such subjects) suchfragments in a vaccine composition, and analyzing the subject(s) for thestimulation of an immune response. Those peptide fragments that inducethe desired response can then be selected.

[0053] Isolated peptidyl portions of IL-13Rα2 proteins can be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, similar to the technique described above, anIL-13Rα2 protein of the present invention may be arbitrarily dividedinto fragments of desired length with no overlap of the fragments, orpreferably divided into overlapping fragments of a desired length. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments which can function antigenicagents that stimulate an immune response against an IL-13Rα2 protein.

[0054] Another aspect of the present invention concerns recombinantforms of the IL-13Rα2 proteins. Recombinant polypeptides preferred foruse in the present invention, in addition to native IL-13Rα2 protein,are encoded by a nucleic acid that has at least 85% sequence identity(e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%)with the nucleic acid sequence of SEQ ID NO:2. In a preferredembodiment, variant IL-13Rα2 have the ability to stimulate an immuneresponse against the native IL-13Rα2 protein.

[0055] IL-13Rα2 protein variants can be generated through varioustechniques known in the art. For example, IL-13Rα2 protein variants canbe made by mutagenesis, such as by introducing discrete pointmutation(s), or by truncation. Mutation can give rise to an IL-13Rα2protein variant having more, substantially the same, or merely a subsetof the antigenic activity of the native IL-13Rα2 protein. Other variantsof IL-13Rα2 that can be generated include those that are resistant ormore susceptible to proteolytic cleavage, as for example, due tomutations which alter protease target sequences. Whether a change in theamino acid sequence of a peptide results in a IL-13Rα2 protein varianthaving greater or lesser antigenic activity than native IL-13Rα2 proteincan be readily determined by comparing the variant with the nativeIL-13Rα2 protein for the ability to stimulate an immune response againstIL-13Rα2 in subjects vaccinated with the respective proteins.

[0056] As another example, IL-13Rα2 protein variants can be generatedfrom a degenerate oligonucleotide sequence. Chemical synthesis of adegenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then ligated into an appropriateexpression vector. The purpose of a degenerate set of genes is toprovide, in one mixture, all of the sequences encoding the desired setof potential IL-13Rα2 protein sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad.Sci. USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409; 5,198,346;and 5,096,815).

[0057] Similarly, a library of coding sequence fragments can be providedfor an IL-13Rα2 gene clone in order to generate a variegated populationof IL-13Rα2 protein fragments for screening and subsequent selection offragments having the ability to stimulate an immune response againstIL-13Rα2 in a subject. A variety of techniques are known in the art forgenerating such libraries, including chemical synthesis. In oneembodiment, a library of coding sequence fragments can be generated by(i) treating a double-stranded PCR fragment of an IL-13Rα2 gene codingsequence with a nuclease under conditions wherein nicking occurs onlyabout once per molecule; (ii) denaturing the double-stranded DNA; (iii)renaturing the DNA to form double-stranded DNA which can includesense/antisense pairs from different nicked products; (iv) removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease; and (v) ligating the resulting fragment library into anexpression vector. By this exemplary method, an expression library canbe derived which codes for N-terminal, C-terminal and internal fragmentsof various sizes.

[0058] The invention also provides for reduction of IL-13Rα2 proteins togenerate mimetics, e.g. peptide or non-peptide agents, that are able tostimulate an immune response against IL-13Rα2 in a subject. Forinstance, non-hydrolyzable peptide analogs of the amino acid residues ofIL-13Rα2 proteins and peptides thereof can be generated usingbenzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gamma lactam rings (Garvey et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986)J. Med. Chem. 29:295; and Ewenson et al. in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), beta-turn dipeptide cores (Nagai etal. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J. Chem. Soc.Perkin. Trans. 1:1231), and b-aminoalcohols (Gordon et al. (1985)Biochem. Biophys. Res. Commun. 126:419; and Dann et al. (1986) Biochem.Biophys. Res. Commun. 134:71). IL-13Ru2 proteins may also be chemicallyZU 2E modified to create IL-13Rα2 derivatives by forming covalent oraggregate conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of IL-13Rα2 proteins or peptides can be prepared by linkingthe chemical moieties to functional groups on amino acid side chains ofthe protein/peptide or at the N-terminus or at the C-terminus of theprotein/peptide.

[0059] IL-13Rα2 proteins may also be fused to one or more otherproteins. For example, an IL-13Rα2 protein or immunogenic portionthereof may be fused to another protein that serves as a targetingligand to deliver the IL-13Rα2 protein or portion to a particular targetsite in a subject (e.g., in order to stimulate a local immune responseat that site). For instance, an IL-13Rα2 protein or peptide can be fusedto a mutant IL-13 molecule or anti-IL-13 receptor antibody tospecifically target the IL-13Rα2 protein or peptide to a tumor, e.g., aHGG. Numerous methods of fusing two or more proteins together are knownin the art, e.g., making and expressing a recombinant fusion construct,or using a cross-linking agent to covalently bond the two or moreproteins together to form one molecule. Any suitable for thisapplication might be used in the invention.

[0060] The IL-13Rα2 proteins and peptides of the invention can be madeby known methods. For example, a host cell transfected with a nucleicacid vector directing expression of a nucleotide sequence encoding thesubject proteins or peptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells may beharvested, lysed, and the protein isolated. A recombinant IL-13Rα2protein or peptide can be isolated from host cells using techniquesknown in the art for purifying proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for such protein or peptide.

[0061] For example, after an IL-13Rα2 protein or peptide has beenexpressed in a cell, it can be isolated using immuno-affinitychromatography. For instance, an anti-IL-13Rα2 antibody thatspecifically binds the subject proteins or peptides can be immobilizedon a column chromatography matrix, and the matrix can be used forimmuno-affinity chromatography to purify the proteins or peptides fromcell lysates by standard methods (see, e.g., Ausubel et al., supra).After immuno-affinity chromatography, the proteins or peptides can befurther purified by other standard techniques, e.g., high performanceliquid chromatography (see, e.g., Fisher, Laboratory Techniques InBiochemistry And Molecular Biology, Work and Burdon, eds., Elsevier,1980). In another embodiment, the IL-13Rα2 proteins or peptides utilizedin the invention are expressed as a fusion protein containing anaffinity tag (e.g., GST) that facilitates its purification.

[0062] In association with an antigenic agent (e.g., a IL-13Rα2 proteinor peptide fragment thereof) of a vaccine of the invention, an adjuvantcan be used to boost the immune response. Suitable adjuvants for use inthe invention can include any substance that can non-specificallyenhance an antigen-specific immune response stimulated by an antigenicagent. Many such adjuvants are known, including for example: (1)Freund's adjuvant (complete and incomplete) (2) oil-in-water emulsionformulations such as the Ribi™ adjuvant system (Corixa, Seattle, Wash.)(3) aluminum salts (e.g., aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc); (4) saponin-based adjuvants (Stimulon™ fromAquila Biosciences, Framingham, Mass.); (5) cytokines such as IL-1,IL-2, macrophage colony stimulating factor, and tumor necrosis factor;and (6) other substances that act as immunostimulating agents such asmuramyl peptides or bacterial cell wall components, toxins, and toxoids.

[0063] To facilitate their formulation for administration to a subject,the vaccine compositions of the invention (e.g., the protein/peptideantigen and adjuvant) can further contain a pharmaceutically acceptablecarrier or excipient. For example the protein/peptide antigen andadjuvant can be mixed with a diluent such as water, saline, glycerol,ethanol, etc. Other substances, such as preservatives, surfactants,emulsifying agents, buffers, etc. can also be included. Typically, theprotein/peptide-based vaccine compositions of the invention are preparedfor parenteral injection as liquid solutions or suspensions. The vaccinecompositions can also be prepared as solids (e.g., a lyophilized powder)that can be reconstituted in a liquid (e.g., saline) prior to injectioninto a subject. The vaccine compositions can also be emulsified orencapsulated in liposomes.

[0064] Nucleic Acid-Based Vaccines

[0065] Nucleic acid-based vaccines are known to elicit a prominentcell-mediated immune response. See, e.g., Donnely et al., 1997;Rosenberg, S. A., Immunity 10:281, 1999. Thus, in addition toprotein/peptide based vaccines, the antigenic agent for use in thevaccines of the invention can take the form of a nucleic acid that canstimulate an immune response against IL-13Rα2 when administered to asubject. Examples of such nucleic acids include those that encode thenative IL-13Rα2 such as the nucleic acid shown herein as SEQ ID NO:2(FIG. 2), a variant of the native IL-13Rα2, or a peptide fragment ofthat native or variant IL-13Rα2. Vaccines made with a nucleic acid thatencodes the whole protein antigen are advantageous because they have thepotential for stimulating an immune response against all of thedifferent antigenic sites expressed by the protein. Vaccines made with anucleic acid that encodes a peptide antigen (e.g., 7-15 amino acids ofthe whole protein), on the other hand, will generally stimulate animmune response against fewer than all of the potential antigenic sitesexpressed by the protein.

[0066] The form of the nucleic acid used in a vaccine of the inventioncan be any suitable for stimulating an immune response against IL-13Rα2when administered to a subject. For example, the nucleic acid can be inthe form of “naked DNA” or it can be incorporated in an expressionvector. A description of suitable nucleic acids is presented below.Nucleic acids that are most immunogenic in a subject can be determinedby preparing several of the below listed nucleic acids (e.g., those thatencode the whole antigen or peptide fragments thereof), administeringthe subject (or a series of genetically similar such subjects) suchnucleic acids in a vaccine composition (e.g., as naked nucleic acid orin an expression vector in a suitable carrier), and analyzing thesubject(s) for the stimulation of an immune response. Those nucleicacids that induce the desired response can then be selected.

[0067] Nucleic acid molecules utilized in the present invention as anantigenic agent may be in the form of RNA or in the form of DNA (e.g.,cDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded orsingle-stranded, and if single-stranded may be the coding (sense) strandor non-coding (anti-sense) strand. The coding sequence which encodes thenative IL-13Rα2 protein may be identical to the nucleotide sequenceshown in FIG. 2. It may also be a different coding sequence which, as aresult of the redundancy or degeneracy of the genetic code, encodes thesame polypeptide as shown in SEQ ID NO:1 (FIG. 1).

[0068] Other nucleic acid molecules useful in the invention are variantsof the native IL-13Rα2 gene such as those that encode fragments (e.g.,post-translationally processed forms of), analogs and derivatives of anative IL-13Rα2 protein. Such variants may be, e.g., a naturallyoccurring allelic variant of the native IL-13Rα2 gene, a homolog of thenative IL-13Rα2 gene, or a non-naturally occurring variant of the nativeIL-13Rα2 gene. These variants have a nucleotide sequence that differsfrom the native IL-13Rα2 gene in one or more bases. For example, thenucleotide sequence of such variants can feature a deletion, addition,or substitution of one or more nucleotides of the native IL-13Rα2 gene.Nucleic acid insertions are preferably of about 1 to 10 contiguousnucleotides, and deletions are preferably of about 1 to 30 contiguousnucleotides.

[0069] Naturally occurring allelic variants of the native IL-13Rα2 genewithin the invention are nucleic acids isolated from human tissue thathave at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,and 99%) sequence identity with the native IL-13Rα2 gene, and encodepolypeptides having structural similarity to native IL-13Rα2 protein.Homologs of the native IL-13Rα2 gene within the invention are nucleicacids isolated from other species that have at least 75% (e.g., 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity withthe native IL-13Rα2 gene, and encode polypeptides having structuralsimilarity to native IL-13Rα2 protein. Public and/or proprietary nucleicacid databases can be searched in an attempt to identify other nucleicacid molecules having a high percent (e.g., 70, 80, 90% or more)sequence identity to the native IL-13Rα2 gene.

[0070] Non-naturally occurring IL-13Rα2 gene variants are nucleic acidsthat do not occur in nature (e.g., are made by the hand of man), have atleast 75% (e.g., 76%, 77%, 78%, 79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%, 96%,97%, 98%, and 99%) sequence identity with the native IL-13Rα2 gene, andencode polypeptides having structural similarity to native IL-13Rα2protein. Examples of non-naturally occurring IL-13Rα2 gene variants arethose that encode a fragment of a IL-13Rα2 protein, those that hybridizeto the native IL-13Rα2 gene or a complement of to the native IL-13Rα2gene under stringent conditions, those that share at least 65% sequenceidentity with the native IL-13Rα2 gene or a complement of the nativeIL-13Rα2 gene, and those that encode a IL-13Rα2 fusion protein.

[0071] Nucleic acids encoding fragments of native IL-13Rα2 proteinwithin the invention are those that encode, e.g., 2,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,25,30,40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300 or more amino acid residues of the native IL-13Rα2 protein.Shorter oligonucleotides (e.g., those of 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18,19, 20, 30, 50, 100, 125, 150, or 200 base pairs inlength) that encode fragments of the native IL-13Rα2 protein can beused. Nucleic acids encoding fragments of native IL-13Rα2 protein can bemade by enzymatic digestion (e.g., using a restriction enzyme) orchemical degradation of the full length native IL-13Rα2 gene or variantsthereof.

[0072] Nucleic acid molecules encoding IL-13Rα2 fusion proteins are alsowithin the invention. Such nucleic acids can be made by preparing aconstruct (e.g., an expression vector) that expresses a IL-13Rα2 fusionprotein when introduced into a suitable host. For example, such aconstruct can be made by ligating a first polynucleotide encoding anIL-13Rα2 protein fused in frame with a second polynucleotide encodinganother protein (e.g., a detectable label or carrier protein) such thatexpression of the construct in a suitable expression system yields afusion protein. IL-13Rα2 fusion proteins can be used, e.g., to enhancethe immunogenicity of IL-13Rα2 peptides, to facilitate purification ofIL-13Rα2 proteins/peptides, or to track the location of the IL-13Rα2fusion protein after it has been administered to a subject.

[0073] Using the nucleotide sequence of the native IL-13Rα2 gene and theamino acid sequence of a native IL-13Rα2 protein, those skilled in theart can create nucleic acid molecules that have minor variations intheir nucleotide sequences, by, for example, standard nucleic acidmutagenesis techniques or by chemical synthesis. Variant IL-13Rα2nucleic acid molecules can be expressed to produce variant IL-13Rα2proteins.

[0074] Naked Nucleic Acid Vaccines

[0075] The invention provides for the use of naked nucleic acid vaccinesto stimulate an immune response against IL-13Rα2. Representative nakednucleic acid vaccines for use in this method include a DNA encoding oneor more immunogenic portions of IL-13Rα2 along with sufficient other 5′and 3′ elements to direct expression of the foregoing. The use of nakednucleic acids for stimulating both class I and class II restrictedimmune responses against a particular protein is known in the art. See,e.g., Rosenberg, S. A., Immunity 10:281, 1999; Ulmer et al., Science,259:1745, 1993; Donnelly et al., Ann. NY Acad. Sci., 772:40, 1995;Scheurs et al., Cancer res. 58:2509, 1998; Hurpin et al., Vaccine16:208, 1998; Lekutis et al., J. Immunol. 158:4471, 1997; Manickan etal., J. Leukoc. Biol. 61:125, 1997. These methods can be adapted for usein the present invention by using a nucleic acid encoding one or moreimmunogenic portions of IL-13Rα2. Naked nucleic acid vaccines can beadministered to a subject by any suitable technique. For example, nakedDNA encoding a peptide portion of IL-13Rα2 can be injected into musclecells of a subject or naked DNA-coated gold particles can be introducedinto skin cells (to be taken up by dendritic cells) of a subject using agene gun.

[0076] Expression Vector Vaccines

[0077] The invention also provides for the use of expression vectorvaccines to stimulate an immune response against IL-13Rα2. In a typicalapplication of this technique, a nucleic acid encoding one or morepeptide or protein antigens of IL-13Rα2 is incorporated into a vectorthat allows expression of the antigen(s) in a host cell (e.g., a cellinside a subject or administered to a subject). The nucleic acidencoding the antigen(s) is generally be under the operational control ofother sequences contained within the vector such as a promoter sequences(e.g., tissue specific, constitutively active, or inducible) or enhancersequences. The antigen(s) encoded by the vector are expressed when thevector is introduced into a host cell in a subject. After expression,the antigen(s) can associate with an MHC molecule for presentation toimmune system cells such as T lymphocytes, thus stimulating an immuneresponse. See, e.g., Corr et al., J. Exp. Med. 184:1555 (1996).

[0078] Vectors for use in the invention can be any capable of expressingan encoded antigen(s) in a subject. For example, vectors derived frombacterial plasmids and viruses may be used. Representative viral vectorsinclude retroviral, adenoviral, and adeno-associated viral vectors. See,e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein,Springer Verlag, 1999; Gene Therapy Protocols (Methods in MolecularMedicine), ed. P. D. Robbins, Humana Press, 1997; and Retro-vectors forHuman Gene Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.

[0079] Cell-Based Vaccines

[0080] Cell-based vaccines are provided in the invention to stimulate animmune response against IL-13Rα2. In similar approaches using differentcancer-associated antigen, cancer cells isolated from a patient havebeen harbored in vitro and transfected with DNA encoding for immunestimulants, such as cytokines, MHC molecules or co-stimulatorymolecules. The transfected cancer cells were then re-injected to thepatient in order to activate the immune system in order to generate ananti-cancer response. Greten, T. F., and E. M. Jaffee, J. Clin. Oncol.,17: 1047-1060, 1999; Simons et al., Cancer Res., 57: 1537-1546, 1997.

[0081] The invention further provides an isolated cell expressingIL-13Rα2 or a peptide fragment of IL-13Rα2. Cells expressing IL-13Rα2can be isolated from a subject having such cells (e.g., from testis orHGG). Cells that do not express IL-13Rα2 can be made to express thisprotein in a number of different ways. As one example, cells can becultured with IL-13Rα2 or peptide fragments thereof under conditions inwhich fragments of IL-13Rα2 become associated with MHC molecules on thecell surface. Alternatively, cells can be made to express IL-13Rα2 byintroducing a nucleic acid encoding an IL-13Rα2 protein, a peptidefragment of IL-13Rα2, or a variant of the foregoing into the cells, andculturing such cells under conditions that cause the cells to expressthe protein or peptide. Cellular expression of the protein, peptide, orvariant can be monitored by any conventional technique. For example,fluorescently labeled antibodies that specifically bind the protein,peptide, or variant can be used to detect expression of the protein,peptide, or variant on a cell. See, e.g., Kim et al., J. Immunother.20:276, 1997. In addition, Western blotting using antibodies thatspecifically bind the protein, peptide, or variant can be used to detectexpression of the protein, peptide, or variant in lysates of a cell.

[0082] Cell types suitable for stimulating an immune response againstIL-13Rα2 can be prokaryotic or eukaryotic. A number of such cells areknown in the art, so an exhaustive list is not provided herein. Examplesof suitable prokaryotic cells include bacterial cells such as E. coli,B. subtilis, and mycobacteria. Examples of suitable eukaryotic cellsinclude plant, yeast, insect, avian, nematode (e.g., C. elegans), andmammalian cells (e.g., autologous cells from a human patient that are tobe later reintroduced into the patient). These cells can be cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

[0083] Further examples of cells that can be used to stimulate an immuneresponse against IL-13Rα2 include those that express a peptidecomprising a least 7 (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more) contiguous amino acids of SEQ ID NO:1. For instance, anisolated cell expressing a protein having the sequence of SEQ ID NO: 1can be used. Cells into which have been introduced a purified nucleicacid that encodes a peptide comprising a least 7 contiguous amino acidsof SEQ ID NO: 1 might also be used.

[0084] Although any cell that can express IL-13Rα2 protein, a peptidefragment of IL-13Rα2, or a variant of the foregoing can be used tostimulate an immune response in a subject, some are preferred because oftheir particular antigen presentation capabilities. Examples of suchcells include antigen-presenting cells (APCs) such as B lymphocytes,monocytes/macrophages, dendritic cells (DC), and other cells expressingmajor histocompatability complex (MHC) and/or costimulatory molecules.

[0085] Since DC are known to function as particularly strong APCs, theiruse in the cell-based vaccine of the invention is particularlypreferred. See, e.g., Banchereau et al., Ann. Rev. Immunology, 18:767,2000. DC can be made to express an IL-13Rα2 protein, a peptide fragmentof IL-13Rα2, or a variant thereof as described above. For example, DCcan be removed from a subject, contacted with the selected antigen, andthen returned to the subject to stimulate an immune response. To enhancetheir antigen presentation capability, the DC can also be treated withan activating substance such as a cytokines.

[0086] Those cell-based vaccines that are most effective in stimulatingan immune response against IL-13Rα2 in a subject can be determined bypreparing a series of different cell-based vaccine (e.g. thoseexpressing whole antigen or specific peptide fragments of the antigen),administering a subject (or a series of genetically similar subjects)such different vaccines, and analyzing the subject(s) for thestimulation of an immune response. Those vaccines that induce thedesired response can then be selected.

[0087] Anti-Idiotypic Antibody Vaccines

[0088] The invention also contemplates the use of anti-idiotypicantibody vaccines to stimulate an immune response against IL-13Rα2 in asubject. In this method, anti-idiotypic antibodies are prepared thatfeature an internal “image” of one or more immunogenic portions ofIL-13Rα2. See, e.g., U.S. Pat. Nos. 5,053,224; 5,208,146; 5,612,030; and5,925,362. Administration of these anti-idiotypic antibodies in avaccine composition to a subject can stimulate an immune responseagainst the “image” of an immunogenic portion of IL-13Rα2 whichcross-reacts against actual immunogenic portions of IL-13Rα2. As oneexample, polyclonal anti-idiotypic antibodies can be generated byimmunizing a host animal with monoclonal antibodies raised against anepitope of IL-13Rα2. Methods of preparing monoclonal and polyclonalantibodies as described in more detail below.

[0089] Antibody Production

[0090] The vaccines/antigenic agents featured in the invention can beused to raise antibodies useful in the invention. Polyclonal antibodiesare heterogeneous populations of antibody molecules that are containedin the sera of the immunized animals. Antibodies within the inventiontherefore include polyclonal antibodies and, in addition, monoclonalantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,and molecules produced using a Fab expression library. Monoclonalantibodies, which are homogeneous populations of antibodies to aparticular antigen, can be prepared using the IL-13Rα2 proteins andpeptides described above and standard hybridoma technology (see, forexample, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J.Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976;Hammerling et al., “Monoclonal Antibodies and T Cell Hybridomas,”Elsevier, N.Y., 1981; Ausubel et al., supra). In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described in Kohler et al., Nature 256:495, 1975, and U.S. Pat.No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA80:2026, 1983), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96, 1983). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridomaproducing a ′nAb of the invention may be cultivated in vitro or in vivo.The ability to produce high titers of rmabs in vivo makes this aparticularly useful method of production.

[0091] Human or humanoid antibodies that specifically bind a IL-13Rα2protein can also be produced using known methods. For example,polyclonal antibodies can also be collected from human subjects havingsuch antibodies in their sera, e.g., subjects administered vaccines thatstimulate antibody production against IL-13Rα2. As another example,human antibodies against IL-13Rα2 protein can be made by adapting knowntechniques for producing human antibodies in animals such as mice. See,e.g., Fishwild, D. M. et al., Nature Biotechnology 14 (1996): 845-851;Heijnen, I. et al., Journal of Clinical Investigation 97 (1996):331-338; Lonberg, N. et al., Nature 368 (1994): 856-859; Morrison, S.L., Nature 368 (1994): 812-813; Neuberger, M., Nature Biotechnology 14(1996): 826; and U.S. Pat. Nos. 5,545,806; 5,569,825; 5,877,397;5,939,598; 6,075,181; 6,091,001; 6,114,598; and 6,130,314. Humanoidantibodies against IL-13Rα2 can be made from non-human antibodies byadapting known methods such as those described in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; and 5,693,762.

[0092] Once produced, polyclonal or monoclonal antibodies can be testedfor specific IL-13Rα2 recognition by Western blot or immunoprecipitationanalysis by standard methods, for example, as described in Ausubel etal., supra. Antibodies that specifically recognize and bind to IL-13Rα2are useful in the invention. For example, such antibodies can be used inan immunoassay to monitor the level of IL-13Rα2 in a sample (e.g., todetermine the amount of cellular expression or subcellular location ofIL-13Rα2, or the presence and amount of soluble forms of IL-13Rα2 in aliquid sample).

[0093] Preferably, IL-13Rα2 protein selective antibodies of theinvention are produced using fragments of the IL-13Rα2 protein that lieoutside highly conserved regions and appear likely to be antigenic bycriteria such as high frequency of charged residues. Cross-reactiveanti-IL-13Rα2 protein antibodies are produced using a fragment of aIL-13Rα2 protein that is conserved among members of this family ofproteins. In one specific example, such fragments are generated bystandard techniques of PCR, and are then cloned into the pGEX expressionvector (Ausubel et al., supra). Fusion proteins are expressed in E. coliand purified using a glutathione agarose affinity matrix as described inAusubel, et al., supra.

[0094] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusions can be generated for each protein,and each fusion can be injected into at least two rabbits. Antisera canbe raised by injections in a series, preferably including at least threebooster injections. Antiserum is also checked for its ability toimmunoprecipitate recombinant IL-13Rα2 proteins or control proteins,such as glucocorticoid receptor, CAT, or luciferase.

[0095] Techniques described for the production of single chainantibodies (e.g., U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692)can be adapted to produce single chain antibodies against a IL-13Rα2protein, or a fragment thereof. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0096] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0097] Method of Inducing an Anti-IL-13Rα2 Immune Response in a Subject

[0098] The invention provides methods for stimulating a immune responseagainst IL-13Rα2 in a subject having or at risk for developing a cancerhaving cells expressing IL-13Rα2. Such methods can be performed by (a)formulating as anti-cancer vaccine composition (as described above)outside of the subject and (b) administering the vaccine to the subjectin an amount sufficient to stimulate an immune response against IL-13Rα2in the subject.

[0099] Subjects

[0100] The compositions and methods of the invention can be utilizedwith any suitable subject, e.g., an animal such as a mammal (e.g., humanbeings, dogs, cats, goats, sheep, cows, horses, etc.). A human patientsuffering or at risk for developing a cancer or other disease that hascells that overexpress IL-13Rα2 (e.g., a brain cancer such as HGG) is aparticularly preferred subject.

[0101] IL-13Rα2 as a Component of a Polyvalent Vaccine

[0102] The invention also provides polyvalent vaccines that incorporateone or more of the foregoing compositions that can stimulate an immuneresponse against IL-13Rα2 in a subject. Two general types of polyvalentvaccines are within the invention. First, a vaccine that contains morethan one agent that can stimulate and immune response against IL-13Rα2(e.g., a composition that contains 2, 3, 4, 5, 6 , 7, 8, or moredifferent peptides listed in Table 1 below). Second, a vaccine thatcontains both (a) an agent that can stimulate and immune responseagainst IL-13Rα2 and (b) a different agent that can stimulate an immuneresponse against a molecule other than IL-13Rα2 (e.g., another TSA orTAA).

[0103] Administering Vaccines to a Subject

[0104] The vaccine compositions of the present invention can be used ina method for stimulating an immune response against IL-13Rα2 in asubject. In this method, an vaccine compositon of the invention can beadministered to a subject by any method that stimulates the aforesaidimmune response. The exact method selected is determined by theparticular vaccine composition to administered. For parenteraladministration by injection, the injection can be in situ (i.e., to aparticular tissue or location on a tissue, e.g., into a tumor or lymphnode), intramuscular, intravenous, intraperitoneal, or by anotherparenteral route. For example, for a protein/peptide based vaccine thevaccine may be administered by subcutaneous or intradermal injection. Insome cases other routes can be used, e.g. intravenous injection,intraperitoneal injection, or in situ injection into target tissue.

[0105] Naked nucleic acid vaccines or expression vector vaccines may beadministered by intramuscular injection. Cell-based vaccines can beintroduced into an animal by any suitable method, e.g., subcutaneousinjection. In addition to parenteral routes, the vaccines of theinvention can also be administered by a non-parenteral route, e.g, byoral, buccal, urethral, vaginal, or rectal administration.

[0106] Formulations for injection may be presented in unit dosage form,for example, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the vaccine compositions may be in powder form(e.g., lyophilized) for constitution with a suitable vehicle, forexample, sterile pyrogen-free water, before use.

[0107] To facilitate delivery of the antigenic compositions (e.g.,antigenic agent plus adjuvant) of the invention to an animal, theantigenic compositions can be mixed with a pharmaceutically acceptablecarrier or excipient. Examples of such pharmaceutically acceptablecarriers and excipients include diluents such as water, saline, citratebuffered saline, phosphate buffered saline, acetate buffered saline, andbicarbonate buffered saline; and stabilizing agents such as amino acids,alcohols, proteins (for example, serum albumin), EDTA, mannitol,sorbitol, and glycerol. To minimize the chance of infection or adversereaction when administered to a subject, carriers and excipients arepreferably sterile and pyrogen-free. USP grade carriers and excipientsare particularly preferred for delivery of vaccine compositions to humansubjects. The vaccine compositions can also be formulated for long-termrelease as a depot preparation by adding the antigenic agent to suitablepolymeric or hydrophobic materials or ion exchange resins. They can alsobe made by preparing the vaccine composition as a sparingly solublederivative. Depot preparations can be administered to a subject byimplantation (e.g., subcutaneous or intramuscular surgical implantation)or by injection. Methods for making the foregoing formulations are wellknown and can be found in, for example, Remington's PharmaceuticalSciences.

[0108] Dosing

[0109] The vaccine compositions of the invention are preferablyadministered to a subject in an amount sufficient to stimulate an immuneresponse against IL-13Rα2 in the subject, and not cause an overly toxiceffect. Such a therapeutically effective amount can be determined asdescribed below.

[0110] Toxicity and therapeutic efficacy of the vaccines utilized in theinvention can be determined by standard pharmaceutical procedures, usingeither cells in culture or experimental animals to determine the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Vaccines that exhibit largetherapeutic indices are preferred. While those that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat minimizes the potential damage of such side effects. Data obtainedfrom animal studies can be used in formulating a range of dosage for usein humans. The dosage of such vaccines lies preferably within a rangethat include an ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized.

[0111] The vaccines of the invention can be administered to a subjectusing various different vaccination schedules. For example, a nucleicacid vaccine might be administered to a subject only once, while aprotein/peptide- based vaccine might be administered to the subject onmultiple occasions (1, 2, 3, 4, 5 or more times). For example, in aneffort to stimulate a strong immune response, a first dose of a vaccinecompositions of the invention may be administered to a subject at least24 hours before a second (booster) dose is administered to the subject.

[0112] Kits

[0113] The invention also provides kits for stimulating an immuneresponse against IL-13Rα2 in a subject. Such kits can include acontainer holding one or more of the antigenic agents described above ina pharmaceutically acceptable form. The antigenic agent(s) in thecontainer can be in liquid form (e.g., as a solution) or in solid form(e.g., as a lyophilized or desiccated powder). Where, for example, theantigenic agent is a solid, the kits within the invention can furtherinclude a container holding a pharmaceutically acceptable solution(e.g., sterile saline with or without dextrose) for reconstituting thesolid into a liquid suitable for injection. The kits of the inventioncan further include (a) one or more devices to administer the antigenicagent, e.g., a needle or syringe, a packaged alcohol pad, etc.; and/or(b) printed instructions for using the kit.

EXAMPLES Example 1 IL-13Rα2 Mimics the Biological Features of anHGG-associated receptor for IL-13

[0114] Normal Chinese hamster ovary (CHO) cells were transfected with apcDNA 3.1 plasmid (Invitrogen) containing the full length open readingframe of IL-13Rα2 and positive clones were selected with geneticin. Theexpression of IL-13Rα2 in these clones was tested for their ability tobind ¹²⁵I-labeled IL-13. Selected clones were shown to bind labeledIL-13 independently of IL-4. In addition, labeled IL-13 was displaced byIL-13.E13K, a mutant of IL-13 shown to have a greater affinity for theIL-13 binding protein on HGG than for the shared IL-13/IL-4 receptorfound in a plethora of tissues under a physiological state. Furthermore,these IL-13Rα transfected CHO cells were exposed to anIL-13.E13K-PE38QQR cytotoxin, a fusion protein showing potent dosedependent cytotoxicity on HGG cells. The clones expressing the receptorwere killed in direct proportion to their affinity for IL-13, but notCHO cells alone or CHO cells transfected with an empty plasmid. Inneutralization experiments, an excess of IL-13 prevented the cytotoxiceffect of IL-13.E 13K-PE38QQR. Therefore the only way the toxin,PE38QQR, could have entered and killed the cells was throughreceptor-mediated endocytosis, a process directed through the IL-13portion of the cytotoxin. Use of an IL13.E13K/enhanced green flourescentprotein (EGFP) fusion protein confirmed that this process occurred.Thus, IL-13Rα2 was demonstrated to share properties ascribed to morerestrictive, IL-4 independent, IL-13 binding sites found on HGGs in situand in vitro.

Example 2 Identification of IL-13Rα2 as a Cancer Testis Antigen

[0115] Materials and Methods

[0116] Sources of RNA.

[0117] High-grade glioma cell lines A-172 MG, U-373 MG, U-251 MG andhuman glioblastoma multiforme explant cells (G-48) were grown in culturein appropriate media. Total RNA was extracted from the cells using theacid-guanidium isothiocyanate-phenol-chloroform method. Poly(A)+ RNA wasfurther isolated using the Mini-oligo(dT) Cellulose Spin Column Kit (5prime-3 prime Inc., Boulder, Colo.). 2 μg of Poly (A)+ RNA waselectrophoresed on a 1% agarose formaldehyde gel, transferred to 0.45 μmmagna charge nylon (MSI, Westborough, Mass.) and UV-crosslinked(Stratagene, La Jolla, Calif.). RNA-blotted membranes were alsopurchased from Clontech (Palo Alto, Calif.). Two Multiple TissueExpression (MTETM) Blots (cat #7770-1 and 7775-1;www.clontech.com/mtn/index.html) were analyzed to determine the tissuedistribution of the IL13 binding proteins. Two sets of Human BrainMultiple Tissue Northern (MTNTM) Blots (cat # 7755-1 and 7769-1) wereassayed to confirm the true presence of the transcripts. In addition,two Human Tissue Northern (MTNTM) Blots (cat #7759-1 and 7760-1) wereanalyzed to verify the tissue distribution of the IL-13 Ra2 transcript.

[0118] cDNA Probes.

[0119] cDNA probes were generated either by PCR (IL-13Rα2 and IL13Rα1)or by restriction digest (IL-4Rα=p140). cDNA containing human IL13Rα2was provided by Dr. Pascual Ferrara of Sanofi Recherche. cDNA containinghuman IL-13Rα1 (and also 93 bases of murine IL-13) was provided by Dr.Douglas J. Hilton of The Walter and Eliza Hall Institute of MedicalResearch. Plasmid pHuIL4R/ID was used to obtain a fragment of IL4Rα bythe restriction digest. The fragments were electrophoresed on a 1%agarose gel, excised from the gel and purified using QlAquick GelExtraction Kit (Qiagen Inc., Valencia, Calif.). Actin cDNA was purchasedfrom Clontech Labs.

[0120] The primers for human IL-13Rα2 were as follows: forward5′-AAGATTTGGAAGCTTATGGCTTTCGTTTGC-3′ (SEQ ID NO:3) reverse5′-TCCCTCGAAGCTTCATGTATCACAGAAAAA-3′ (SEQ ID NO:4)

[0121] The primers for human IL13Rα1 were as follows: forward5′-ATTATTAAGCTTATGGAGTGGCCGGCG-3′ (SEQ ID NO:5) reverse5′-TAACCGGAAGCTTCACTGAGAGGCTTT-3′ (SEQ ID NO:6)

[0122] Northern Blot Analysis.

[0123] Membranes were pre-hybridized overnight at 42° C. in a solutionconsisting of 50% formamide, 5× SSC, 50 mM sodium phosphate, 5×Denhardt's, 50 μg/ml sheared salmon sperm DNA, and 1% SDS. Membraneswere subsequently hybridized overnight at 42° C. in the same solutionwith the addition of full length cDNA probes labeled by random priming(Life Technologies, Rockville, Md.) with ³²P-dCTP using 1-2×10⁶ cpm/ml.Following hybridization, the membranes were washed with 2×SSC/0.2% SDSat 42° C. for 20 minutes followed by two washes with 1× SSC/0.1% SDS at42° C. for 20 minutes each. The membranes were exposed toautoradioraphic film X-OMAT AR (Eastman Kodak Co., Rochester, N.Y.) andplaced at −80° C. for 1, 3 and 14 days. The membranes were subsequentlystripped and re-probed up to three more times. The membranes were probedfirst with IL-13Rα2, followed by IL13Rα1, IL-4Rα=p140, and actin. Filmswere scanned on a transparency scanner at a pixel size of 88×88 micron(Molecular Dynamics, Sunnyvale, Calif.). The images were compiled inPaint Shop Pro V 5.0 (Jasc software Inc., Eden Prairie, Minn.).

[0124] Results

[0125] Northern blot analysis of transcripts for IL-13Rα2 in normalorgans. To explore the expression of IL-13Rα2, an extensive examinationof the presence of transcripts for this protein among multiple normaltissues, including 20 discrete regions of the CNS and a variety ofnormal peripheral organs was performed. All Northern blots using samemembranes were performed with respective labeled cDNAs in the followingorder: IL-13Rα2, ILI3Rα1, IL4α and β-actin. This assured that the levelsof transcripts for IL-13Rα2 were not underestimated due to the usage ofmembranes with mRNA. Both the dot-blot analyses (not shown) and theelectrophoretically separated transcripts for IL-13Rα2 (FIG. 4, panelsI-IV) demonstrated mostly undetectable, or very weak signals in fewcases, of IL-13Rα2 transcripts in the organs studied, even after 2-weekof film exposure. The first dot blot performed, however, surprisinglyshowed an unusually high density of labeling with IL-13Rα2 cDNA probe toto transcripts derived from testis. This was also found using anotherNorthern blot membrane. A few other organs had transcripts thathybridized to the IL-13Rα2 cDNA (aorta, liver, and pituitary gland). Thedensity of labeling in the dot blots was much lower than in the testisblot. Of importance, there was no evidence for the presence ofsignificant IL-13Rα2 expression in the CNS.

[0126] To confirm these findings made using dot blot analysis,additional blots were performed using electrophoretically separatedmRNAs. Again, the discrete regions of normal human brain did not produceclear-cut hybridization signals (FIG. 4, panels I and II). On the otherhand, the only organ with prominent hybridization band corresponding tothe mRNA of 1.5 kb was seen in testis (FIG. 4, panel III). Poorlydetectable signals were seen in placenta, liver, and kidney (FIG. 4,panel IV). Thus, among normal tissues, testes was the only one thatprominently expressed IL-13Rα2. No transcripts for IL-13Rα2 were readilydetected in the CNS.

[0127] Northern blot analysis of transcripts for IL13Rα1 in normaltissues. The expression of IL13Rα1, a component of a heterodimeric formof IL13 receptor that is shared with IL4, IL 13/4 receptor was examinedin a variety of normal human tissues (FIG. 5) by either dot-blotanalyses (not shown) or blots of electrophoretically separatedtranscripts (FIG. 5, panels I-IV). The results unequivocallydemonstrated that IL 13Rα1 was expressed in a variety of the organs,including CNS tissue from medulla, spinal cord, substantia nigra,thalamus, and corpus callosum. Size fractionated mRNAs confirmed themany positive signals seen in dot blots with the strongest signalsobserved in ovary, heart, liver and lung (FIG. 5, panels III and IV,respectively). Of interest, liver showed two hybridized species of MRNA:one of 4.5 kb and the other of 2.0 kb, as an example of a normal organwith doublet of positive signals of different sizes. In summary,discrete regions of normal human brain did produce clear-cut positivehybridization signals for IL 13Rα1 (FIG. 5, panels I and II). Inaddition, many vital peripheral organs exhibited hybridization bandscorresponding to the mRNA of 4.5-4.65 kb (FIG. 5, panels III and IV).

[0128] Gene expression analysis of IL4Rα in normal tissues. In additionto IL13Rα1, IL4Rα is another component of a heterodimeric form of IL13receptor that is shared with IL4, i.e., the shared IL13/4 receptor.Thus, whether the distribution of IL4RU gene expression corresponded tothat of IL13Rα1 was analyzed. All Northern blot analysis membranes usedin this study demonstrated enriched content of the IL4Rα transcripts ina variety of tissues (FIG. 6, panels I, II, and IV). The presence of thetranscripts within the CNS was most evident, as it was for IL13Rα1, inmedulla, spinal cord, substantia nigra and thalamus (FIG. 6, panels Iand II). Among normal peripheral organs, liver, lung, kidney, intestinaltract, spleen, stomach, and testis demonstrated gene expression ofIL4Rα, which was generally similar to that seen with IL13Rα1 (notshown). Thus, discrete regions of normal human brain contain transcriptsfor both IL13Rα1 and IL4Rα, a complete heterodimer of the shared IL13/4receptor. Furthermore, several vital peripheral organs contained the twosubunits of the IL13/4 receptor, including heart, liver, lung andintestinal tract.

[0129] Control Hybridization of β-Actin.

[0130] All membranes used for Northern blot analysis of IL13 receptorstranscripts were also hybridized with a cDNA probe for a house-keepinggene, β-actin (FIG. 7; dot blots and panel III not shown). The intensityof the signals for β-actin was usually in accordance with the amount ofmRNA present on the membranes, as estimated by the manufacturer.

[0131] Gene Expression of IL 13Receptors in Cells.

[0132] Gene expression of the two IL13 receptors was also examined inmalignant and normal cells (FIG. 8). Transcripts for IL13Rα2, IL13Rα1,IL4Rα and β-actin were examined in serial hybridization assays. Isolatedexplant cells of HGG (G-48) as well as human malignant gliomaestablished cell lines (A-172 MG, U-373 MG, and U-251 MG) demonstratedintense signals for IL-13Rα2 (FIG. 8). On the other hand, thetranscripts for the elements of the shared IL 13/4 receptor, IL-13Rα1and IL4Rα, were found at lower levels when compared with that forIL-13Rα2 (FIG. 8). A-172 MG cells appeared to be the most enriched inthe components of the IL 13/4 receptor heterodimer. Of interest, twospecies of different sizes of the transcripts for both IL-13Rα2 andIL-13Rα1 were seen in cells (FIG. 8). In a control assay, humanumbilical vein endothelial cells (HUVEC) showed the presence oftranscripts for IL-13Rα1 and IL4Rα, but not those for IL-13Rα2 (FIG. 8).In summary, gene expression of IL-13Rα2 was detected in two specimens ofHGG (FIG. 8, HGG 13 and HGG 52), but not in two normal brain specimens(FIG. 8, NB 3 and NB 6). However, the transcripts for IL-13Rα1 werefound in all of these specimens. In other experiments, severaladditional HGG brain tumor specimens were determined to expressIL-13Rα2.

Example 3 Representative Immunogenic Peptides of IL-13Rα2

[0133] Table I presents a list of IL-13Rα2 peptides that might be usedto stimulate an immune response against IL-13Rα2 in a subject. Thelisted peptides were obtained using a computer program provided by theLudwig Institute For Cancer Research (Lausanne, Switzerland) on theInternet at http://www-ludwig.unil.ch.SEREX.html. This program providedthe best (at high stingency) fit of predicted immunogenic peptides thatbind specific classes of MHC molecules (i.e., the various alleles ofhuman MHC Class I indicated in Table I). The peptides indicated with the“*” are those that should bind under high stringency. The skilledartisan could produce these peptides as described herein (e.g., byautomated peptide synthesis) and use each in a vaccine preparation thatwould be administered to a variety of test subjects (e.g. those withdifferent MHC types) as also described herein. The immune responsestimulated by each of these peptides in the subjects could then beassessed, so that those that stimulate the desired immune responses inparticular test subjects could be identified. TABLE I Binding peptidesprediction: Allele Peptide Position Score t½ A1 IVDP-GYLGY 16-24 7.1201236.45043346563 A1 LLDTNYNLFY 140-149 4.820 123.965090779824 A_0201YLYLQWQPPL * 24-33   5.760 317.34832891785 A_0201 YLQWQPPLSL * 26-35  4.600 99.4843156419338 A_0201 LQWQ-PPLSL 27-35 3.430 30.876642749677A_0201 SLDHFKECTV 34-43 3.330 27.9383417032365 A_0201 NLHYKDGFDL * 64-73  4.830 125.210960654765 A_0201 WQCT-NGSEV 87-95 3.490 32.7859477062319A_0201 CVYY-NWQYL * 121-129   4.020 55.7011058267956 A_0201 YLLCSWKPGI *128-137   5.190 179.468552931832 A_0201 VLLD-TNYNL * 139-147   6.320555.572992451403 A_0201 NLFY-WYEGL * 146-154   4.080 59.1454698498823A_0201 GLDH-ALQCV * 153-161   4.160 64.0715225999366 A_0201 NIGC-RFPYL170-178 3.420 30.5694150210502 A_0201 FQLQNIVKPL * 206-215   4.45085.6269440022006 A_0201 QLQN-IVKPL * 207-215   3.900 49.4024491055302A_0201 NIVK-PLPPV 210-218 3.090 21.9770779757634 A_0201 YLTFTRESSC219-228 3.140 23.1038668587222 A_0201 QLCFVVRSKV * 279-288   4.25070.1054123466879 A_0205 IVDPGYLGYL 16-25 3.120 22.6463796431754 A_0205YLYLQWQPPL * 24-33   4.140 62.8028214492017 A_0205 LQWQ-PPLSL 27-353.350 28.5027336437673 A_0205 LQWQ-PPLSL 26-35 3.040 20.9052432350928A_0205 CVYY-NWQYL * 121-129   4.430 83.9314169102688 A_0205 VLLD-TNYNL *139-147   4.670 106.697742432451 A_0205 VLLD-TNYNL * 138-147   3.74042.0979901649969 A_0205 NLFY-WYEGL 146-154 3.040 20.9052432350928 A_0205FQLQNIVKPL * 206-215   4.610 100.484149636389 A3 LLDTNYNLFY 140-1493.190 24.2884274430946 A3 ALQC-VDYIK 157-165 4.520 91.8355979781567 A3GIWS-EWSDK 296-304 3.410 30.2652442594001 A24 DFEIVDPGYL 13-22 3.41030.2652442594001 A24 LYLQ-WQPPL * 25-33   5.710 301.87106828279 A24EYEL-KYRNI * 44-52   4.320 75.1886282920231 A24 TYWI-SPQGI * 103-111  4.090 59.7398917041452 A24 VYYN-WQYLL * 122-130   5.300 200.336809974792A24 WYEG-LDHAL * 150-158   5.890 361.405284372286 A24 DYIKADGQNI *162-171   4.500 90.0171313005218 A24 SYFTFQLQNI * 202-211   4.09059.7398917041452 A DLSK-KTLLR 311-319 3.300 27.1126389206579 A68.1TVEY-ELKYR * 42-50   5.300 200.336809974792 A68.1 TVEY-ELKYR * 41-50  4.600 99.4843156419338 A68.1 ETWK-TILTK * 55-63   4.500 90.0171313005218A68.1 CVNG-SSENK * 189-197   4.790 120.301368663215 A68.1 FTFQLQN1VK *204-213   4.090 59.7398917041452 A68.1 FTRESSCEIK 222-231 3.40029.964100047397 A68.1 ESSC-EIKLK 225-233 3.300 27.1126389206579 A68.1TVENETYTLK * 263-272   4.790 120.301368663215 A68.1 YTLKTPNETR * 269-278  4.600 99.4843156419338 A68.1 ETRQLCFVVR * 276-285   5.010149.904736149047 B7 DPGYLGYLYL 18-27 4.390 80.640418980477 B7 CVYY-NWQYL121-129 3.000 20.0855369231877 B7 GVLLDTNYNL 138-147 3.00020.0855369231877 B7 IVKPLPPVYL 211-220 3.410 30.2652442594001 B7EIRE-DDTTL 251-259 3.690 40.0448469572867 B8_8mer EAKIHTLL 78-85 3.47032.1367424447532 B8_8mer EIKLKWSI 229-236 3.690 40.0448469572867 B8_8merVVRSKVNI 283-290 3.000 20.0855369231877 B14 QNIGCRFPYL 169-178 3.40029.964100047397 B14 IRSSYFTFQL 199-208 3.000 20.0855369231877 B_2702LQWQ-PPLSL 27-35 3.410 30.2652442594001 B_2702 WQPPLSLDHF 29-38 3.00020.0855369231877 B_2702 YRNI-GSETW 49-57 4.610 100.484149636389 B_2702VQSSWAETTY 95-104 3.000 20.0855369231877 B_2702 VQDM-DCVYY 116-124 3.00020.0855369231877 B_2702 GQNIGCRFPY 168-177 3.000 20.085536923 1877B_2702 CRPP-YLEAS 173-181 3.920 50.4004447780655 B_2702 IRSSYFTFQL199-208 4.100 60.340287597362 B_2702 TRESSCEIKL 223-232 4.10060.340287597362 B_2702 ARCFDYEIEI 243-252 4.100 60.340287597362 B_2702IRED-DJTLV 252-260 3.000 20.085536923 1877 B_2702 VRSK-VNIYC 284-2923.000 20.085536923 1877 B_2705 FEIV-DPGYL 14-22 3.400 29.964 100047397B_2705 YLYLQWQPPL 24-33 5.010 149.904736149047 B_2705 LQWQ-PPLSL 27-356.910 1002.24724229025 B_2705 LQWQ-PPLSL 26-35 3.400 29.964100047397B_2705 WQPPLSLDHF 29-38 4.610 100.484149636389 B_2705 KECT-VEYEL 39-474.500 90.0171313005218 B_2705 YRNIGSETWK 49-58 7.600 1998.19589510412B_2705 RNIG-SETWK 50-58 4.090 59.7398917041452 B_2705 SETWKTIITK 54-633.400 29.964100047397 B_2705 KNLH-YKDGF 63-71 3.400 29.964100047397B_2705 NLHYKDGFDL 64-73 3.400 29.964100047397 B_2705 IEAK-IHTLL 77-853.400 29.964100047397 B_2705 WQCT-NGSBV 87-95 4.100 60.340287597362B_2705 VQSSWAETTY 95-104 4.610 100.484149636389 B_2705 VQDM-DCVYY116-124 4.610 100.484149636389 B_2705 CVYY-NWQYL 121-129 3.91049.8989519734079 B_2705 WQYL-LCSWK 126-134 6.910 1002.24724229025 B_2705CSWKPGIGVL 131-140 3.910 49.8989519734079 B_2705 VLLD-TNYNL 139-1473.400 29.964100047397 B_2705 TNYN-LFYWY 143-151 3.910 49.8989519734079B_2705 NLFY-WYEGL 146-154 5.010 149.904736149047 B_2705 ALQC-VDYIK157-165 3.400 29.964100047397 B_2705 LQCV-DYIKA 158-166 3.00020.0855369231877 B_2705 GQNIGCRFPY 168-177 4.610 100.484149636389 B_2705CRFP-YLEAS 173-181 6.910 1002.24724229025 B_2705 FPYLEASDYK 175-1843.910 49.8989519734079 B_2705 IRSSYFTFQL 199-208 7.600 1998.19589510412B_2705 RSSY-FTFQL 200-208 3.400 29.964100047397 B_2705 FTFQLQNIVK204-213 3.910 49.8989519734079 B_2705 FQLQNIVKPL 206-215 4.10060.340287597362 B_2705 TRES-SCEIK 223-231 7.600 1998.19589510412 B_2705RESS-CEIKI 224-232 4.500 90.0171313005218 B_2705 ARCFDYEIEI 243-2526.400 601.845037872082 B_2705 RCFDYEIEIR 244-253 4.320 75.1886282920231B_2705 IRED-DTTLV 252-260 6.400 601.845037872082 B_2705 IEIREDDTTL250-259 3.400 29.964 100047397 B_2705 VENE-TYTLK 264-272 3.400 29.964100047397 B_2705 TRQL-CFVVR 277-285 6.910 1002.24724229025 B_2705RQLCFVVRSK 278-287 5.200 181.272241875151 B_2705 VRSK-VNIYC 284-2925.300 200.336809974792 B_2705 GIWS-EWSDS 296-304 3.910 49.8989519734079B_2705 KQCW-EGEDL 304-312 6.400 601.845037872082 B_2705 QCWEGEDLSK305-314 3.910 49.8989519734079 B_2705 WEGE-DLSKK 307-315 3.40029.964100047397 B_2705 GEDLSKKTLL 309-318 3.400 29.964100047397 B_3501DPGY-LGYLY 18-26 3.700 40.4473043600674 B_3501 QPPL-SLDHF 30-38 3.00020.0855369231877 B_3501 FPYL-EASDY 175-183 4.110 60.9467175696222 B_3501KPIRSSYFTF 197-206 3.690 40.0448469572867 B_3501 KPLPPVYLTF 213-2223.690 40.0448469572867 B_3501 GPIPARCFDY 239-248 3.700 40.4473043600674B3501_8mer DPGYLGYL 18-25 3.000 20.0855369231877 B3501_8mer KPGIGVLL134-141 3.690 40.0448469572867 B3501_8mer KPIRSSYF 197-204 3.69040.0448469572867 B3501_8mer KPLPPVYL 213-220 3.690 40.0448469572867B3501_8mer LPPVYLTF 215-222 3.000 20.0855369231877 B3501_8mer GPIPARCF239-246 3.000 20.0855369231877 B3501_8mer IPARCEDY 241-248 3.70040.4473043600674 B_3701 VDPG-YLGYL 17-25 3.690 40.0448469572867 B_3701KDGFDLNKGI 68-77 3.690 40.04484695272867 B_3701 IEAK-IHTLL 77-85 4.32075.1886282920231 B_3701 LDTN-YNLFY 141-149 3.690 40.0448469572867 B_3701EDLS-KKTLL 310-318 5.300 200.336809974792 B_3701 EDLS-KKTLL 309-3183.910 49.8989519734079 B LHYK-DGFDL 65-73 3.400 29.964100047397 B_3901LHYK-DGFDL 65-73 5.190 179.468552931832 B_3901 DHALQCVDYI 155-164 3.81045.1504388663187 B_3901 TRESSCEIKL 223-232 3.120 22.6463796431754 B_3901IRED-DTTLV 252-260 3.400 29.964100047397 B3901_8mer DHFKECTV 36-43 4.09059.7398917041452 B3901_8mer IREDDTTL 252-259 4.500 90.0171313005218B_3902 LQWQ-PPLSL 27-35 3.000 20.0855369231877 B_3902 FKECTVEYEL 38-473.180 24.046753520645 B_3902 WKTI-ITKNEL 57-65 3.180 24.0467535520645B_3902 WKPG-IGVLL 133-141 3.180 24.0467535520645 B_3902 FQLQNIVKPL206-215 3.180 24.0467535520645 B_3902 VKPL-PPVYL 212-220 3.00020.0855369231877 B_3902 IKLK-WSIPL 230-238 3.180 24.0467535520645 B_3902LKTTNETRQL 271-280 3.000 20.0855369231877 B_3902 KQCW-EGEDL 304-3123.000 20.0855369231877 B_3902 DKQCWBGEDLY 303-312 3.000 20.0855369231877B40 FEIV-DPGYL 14-22 4.390 80.640418980477 B40 KECT-VEYEL 39-47 3.00020.0855369231877 B40 IEAK-IHTLL 77-85 3.690 40.0448469572867 B40RESS-CEIKIL 224-232 3.000 20.085536923 1877 B40 IEIREDDTTL 250-259 4.39080.640418980477 B40 SEWS-DKQCW 299-307 3.690 40.0448469572867 B40GEDL-SKKTL 309-317 3.000 20.0855369231877 B_4403 QDFEIVDPGY 12-21 3.12022.6463796431754 B_4403 FEIV-DPGYI 14-22 3.000 20.0855369231877 B_4403VDPGYLGYLY 17-26 3.210 22.6463796431754 B_4403 KTIITKNLHY 58-67 3.53034.1239676147544 B_4403 QNIG-CRFPY 169-177 3.530 34.1239676147544 B_4403LEASDYKDFY 178-187 5.480 239.846707374255 B_4403 SENKPIRSSY 194-2035.480 23 9.846707374255 B_4403 CEIK-LKWSI 228-236 3.000 20.0855369231877B_4403 GPIPARCFDY 239-248 3.810 45.1504388663187 B_4403 YEIEIREDDT248-257 3.000 20.0855369231877 B_4403 IEIREDDTTL 250-259 3.41030.2652442594001 B_4403 SEWS-DKQCW 299-307 3.180 24.0467535520645 B_5101NPPQ-DFEIV 9-17 5.410 223.631587680546 B_5101 DPGYLGYLYL 18-27 5.400221.406416204187 B_5101 IGSE-TWKTI 52-60 5.050 156.022464486395 B_5101DGFD-LNKGI 69-77 6.070 432.680681574476 B_5101 SPQGIPETKV 107-116 5.410223.631587680546 B_5101 IPET-KVQDM 111-119 3.770 43.3800648358516 B_5101EGLDHALQCV 152-161 4.790 120.3013686632215 B_5101 HALQ-CVDYI 156-1645.300 200.336809974792 B_5101 EASDYKDFYI 179-188 6.090 441.421411145971B_5101 NGSS-ENKPI 191-199 4.590 98.4944301619463 B_5101 IPARCFDYEI241-250 6.260 523.218940108001 B_5101 PARC-FDYEI 242-250 3.00020.0855369231877 B_5101 EGEDLSKKTL 308-317 4.190 66.0227909604099B5101_8mer NPPQDFEI 9-16 6.100 445.857770082517 B5101_8mer PPQDFEIV10-17 3.110 22.4210444007463 B5101_8mer DPGYLGYL 18-25 5.300200.336809974792 B5101_8mer EAKIHTLL 78-85 4.700 109.947172452124B5101_8mer WAETTYWI 99-106 5.400 221.406416204187 B5101_8mer QGIPETKV109-116 3.800 44.7011844933008 B5101_8mer KPGIGVLL 134-141 4.12061.5592422644285 BS101_8mer IGCRFPYL 171-178 3.260 26.0495371425183B5101_8mer KPLPPVYL 213-220 3.920 50.4004447780655 B_5102 NPPQ-DFEIV 9-75.510 247.151127067624 B_5102 DPGYLGYLYL 18-27 4.810 122.731617517265B_5102 IGSE-TWKTI 52-60 4.790 120.301368663215 B_5102 DGFD-LNKGI 69-776.200 592.749041093256 B_5102 KGIEAKIHTL 75-84 4.400 81.4508686649681B_5102 LPWQ-CTNGS 85-93 3.430 30.876642749677 B_5102 SSWAETIYWI 97-1063.200 24.5325301971094 B_5102 TYWI-SPQGI 103-111 3.100 22.1979512814416B_5102 TTYWISPOGI 102-111 3.100 22.1979512814416 B_5102 SPQGIPETKV107-116 6.100 445.857770082517 B_5102 YLLCSWKPGI 128-137 3.18024.0467535520645 B_5102 EGLDHALQCV 152-161 4.900 134.289779684936 B_5102HALQ-CVDYI 156-164 6.600 735.095189241973 B_5102 FPYL-EASDY 175-1833.510 33.4482677839449 B_5102 EASDYKDFYI 179-188 5.400 221.406416204187B_5102 NGSS-ENKPI 191-199 4.590 98.4944301619463 B_5102 KPIR-SSYFT197-205 3.510 33.4482677839449 B_5102 SYFTFQLQNI 202-211 3.30027.1126389206579 B_5102 FTFQ-LQNIV 204-212 3.200 24.5325301971094 B_5102KPLP-PVYLT 213-221 3.410 30.2652442594001 B_5102 IPLGPIPARC 236-2454.200 66.6863310409252 B_5102 IPARCFDYEI 241-250 6.100 445.857770082517B_5102 RCFD-YEIEI 244-252 3.000 20.0855369231877 B_5102 FVVR-SKVNI282-290 3.280 26.575772699874 B_5102 LCF-VRSKV 280-288 3.10022.1979512814416 B_5102 NIYC-SDDGI 289-297 3.000 20.0855369231877B5102_8mer NPPQDFEI 9-16 6.200 492.749041093256 B5102_8mer PPQDFEIV10-17 3.010 20.2873999252409 B5102_8mer DPGYLGYL 18-25 4.610100.484149636389 B5102_8mer EAKIHTLL 78-85 3.320 27.6603505585 167B5102_8mer WAETTYWI 99-106 4.810 122.731617517265 B5102_8mer YWISPQGI104-111 3.280 26.575772699874 B5102_8mer QGIPETKV 109-116 5.000148.413159102577 B5102_8mer KPGIGVLL 134-141 4.710 111.052159905699B5102_8mer IGCRIFPYL 171-178 3.100 22.1979512814416 B5102_8mer FTFQLQNI204-211 3.890 48.9108865237319 B5102_8mer KPLPPVYL 213-220 5.710301.87106828279 B5102_8mer IPLGPIPA 236-243 3.610 36.9660528148225B_5103 NPPQ-DFEIV 9-17 3.800 44.7011844933008 B_5103 IGSETWKTII 52-613.900 49.4024491055302 B_5103 DGFD-LNKGI 69-77 3.980 53.5170342274912B_5103 SPQGIPETKV 107-116 3.800 44.7011844933008 B_5103 EGLDHALQCV152-161 3.980 53.5170342274912 B_5103 HALQ-CVDYI 156-164 4.890132.953574051283 B_5103 EASDYKDFYI 179-188 4.610 100.484149636389 B_5103NGSS-ENKPI 191-199 3.700 40.4473043600674 B_5103 IPARCFDYEI 241-2503.800 44.7011844933008 B_5201 NPPQ-DFEIV 9-17 4.700 109.947172452124B_5201 NPPQ-DFEIV 8-17 3.680 39.6463940725726 B_5201 IGSETWKTII 52-614.600 99.4843156419338 B_5201 DGFD-LNKGI 69-77 4.110 60.9467175696222B_5201 FTFQ-LQNIV 204-212 4.600 99.4843156419338 B_5801 KTIITKNLHY 58-673.000 20.0855369231877 B_5801 SSWA-ETTYW 97-105 4.390 80.640418980477B_5801 QSSWAETTYW 96-105 4.390 80.640418980477 B_5801 DTNY-NLFYW 142-1503.370 29.0785270577971 B_5801 KPLPPVYLTF 213-222 3.100 22.1979512814416B_5801 SSCE-IKLKW 226-234 5.690 295.893620640484 B_5801 SSCE-IKLKW225-234 3.800 44.7011844933008 B_5801 TTNETRQLCF 273-282 4.49089.1214458786587 B_5801 CSDDGIWSEW 292-301 4.900 134.289779684936 B_5801WSEWSDKQCW 298-307 4.390 80.640418980477 B60 FEIV-DPGYL 14-22 5.770320.537732647356 B60 VDPG-YLGYL 17-25 3.000 20.085536923 1877 B60KECT-VEYEL 39-47 5.870 3 54.248980267765 B60 IEAK-IHTLL 77-85 5.870354.248980267765 B60 RESS-CELKL 224-232 6.560 706.271694595366 B60IEIREDDTTL 250-259 5.770 320.537732647356 B60 GEDL-SKKTL 309-317 5.080160.774055928607 B60 EDLS-KKTLL 310-318 3.690 40.0448469572867 B61REDDTTLVTA 253-262 3.100 22.1979512814416 B61 NETR-QLCFV 275-283 4.38079.8380334050845 B61_8mer SEVQSSWA 93-100 3.690 40.0448469572867B61_8mer REDDTTLV 253-260 3.790 44.2564002759834 Cw_0301 FEIV-DPGYL14-22 3.000 20.0855369231877 Cw_0301 LYLQ-WQPPL 25-33 3.00020.0855369231877 Cw_0301 YLYLQWQPPL 24-33 3.000 20.0855369231877 Cw_0301VEYELKYRNI 43-52 3.630 37.7128166171817 Cw_0301 LHYK-DGFDL 65-73 3.00020.0855369231877 Cw_0301 KGIEAKIHTL 75-84 3.590 36.2340759264765 Cw_0301CVYY-NWQYL 121-129 3.360 28.7891908792427 Cw_0301 DCVYYNWQYL 120-1293.360 28.7891908792427 Cw_0301 VYYN-WQYLL 122-130 3.000 20.0855369231877Cw_0301 VLLDTNYNLF 139-148 3.400 29.964100047397 Cw_0301 GVLLDTNYNL138-147 3.000 20.0855369231877 Cw_0301 YNLFYWYEGL 145-154 3.610100.484149636389 Cw_0301 NLFY-WYEGL 146-154 3.410 30.2652442594001Cw_0301 QNIGCRFPYL 169-178 3.610 100.484149636389 Cw_0301 KPIRSSYFTF197-206 3.810 45.1504388663187 Cw_0301 FQLQNIVKPL 206-215 3.18024.0467535520645 Cw_0301 KPLPPVYLTF 213-222 5.010 149.904736149047Cw_0301 IKLK-WSIPL 230-238 3.000 20.0855369231877 Cw_0301 ATVENETYTL262-271 3.590 36.2340759264765 Cw_0401 DFEIVDPGYL 13-22 5.300200.336809974792 Cw_0401 DPGYLGYLYL 18-27 4.390 80.640418980477 Cw_0401LYLQ-WQPPL 25-33 5.300 200.336809974792 Cw_0401 QPPL-SLDHF 30-38 4.49089.1214458786587 Cw_0401 HFKE-CTVEY 37-45 3.400 29.964100047397 Cw_0401EYEL-KYRNI 44-52 3.220 25.0281201813378 Cw_0401 TWKKTIITKNL 56-65 3.69040.0448469572867 Cw_0401 TYWI-SPQGI 103-111 3.220 25.0281201813378Cw_0401 IPET-KVQDM 111-199 4.390 80.640418980477 Cw_0401 VYYN-WQYLL122-130 5.300 200.336809974792 Cw_0401 SWKP-GIGVL 132-140 4.56095.5834798300662 Cw_0401 WYEG-LDHAL 150-158 5.300 200.336809974792Cw_0401 WYEG-LDHAL 149-158 3.870 47.9423860808193 Cw_0401 DYIKADGQNI162-171 3.220 25.0281201813378 Cw_0401 RFPYLEASDY 174-183 3.22025.0281201813378 Cw_0401 DYKD-FYICV 182-190 3.400 29.964100047397Cw_0401 KPIRSSYFTF 197-206 3.700 40.4473043600674 Cw_0401 YFTF-QLQNI203-211 3.910 49.8989519734079 Cw_0401 SYFTFQLQNI 202-211 3.91049.8989519734079 Cw_0401 KPLPPVYLTF 213-222 3.880 48.4242150713452Cw_0401 TFTRESSCEI 221-230 3.220 25.0281201813378 Cw_0401 CFVVRSKVNI281-290 3.220 25.0281201813378 Cw_0702 DPGY-LGYLY 18-26 3.87047.9423860808193 Cw_0702 DPGY-LGYLY 17-26 3.460 31.8169765146677

[0134] Other Embodiments

[0135] This description has been by way of example of how thecompositions and methods of invention can be made and carried out. Thoseof ordinary skill in the art will recognize that various details may bemodified in arriving at the other detailed embodiments, and that many ofthese embodiments will come within the scope of the invention.Therefore, to apprise the public of the scope of the invention and theembodiments covered by the invention, the following claims are made.

1 6 1 380 PRT Homo sapiens 1 Met Ala Phe Val Cys Leu Ala Ile Gly Cys LeuTyr Thr Phe Leu Ile 1 5 10 15 Ser Thr Thr Phe Gly Cys Thr Ser Ser SerAsp Thr Glu Ile Lys Val 20 25 30 Asn Pro Pro Gln Asp Phe Glu Ile Val AspPro Gly Tyr Leu Gly Tyr 35 40 45 Leu Tyr Leu Gln Trp Gln Pro Pro Leu SerLeu Asp His Phe Lys Glu 50 55 60 Cys Thr Val Glu Tyr Glu Leu Lys Tyr ArgAsn Ile Gly Ser Glu Thr 65 70 75 80 Trp Lys Thr Ile Ile Thr Lys Asn LeuHis Tyr Lys Asp Gly Phe Asp 85 90 95 Leu Asn Lys Gly Ile Glu Ala Lys IleHis Thr Leu Leu Pro Trp Gln 100 105 110 Cys Thr Asn Gly Ser Glu Val GlnSer Ser Trp Ala Glu Thr Thr Tyr 115 120 125 Trp Ile Ser Pro Gln Gly IlePro Glu Thr Lys Val Gln Asp Met Asp 130 135 140 Cys Val Tyr Tyr Asn TrpGln Tyr Leu Leu Cys Ser Trp Lys Pro Gly 145 150 155 160 Ile Gly Val LeuLeu Asp Thr Asn Tyr Asn Leu Phe Tyr Trp Tyr Glu 165 170 175 Gly Leu AspHis Ala Leu Gln Cys Val Asp Tyr Ile Lys Ala Asp Gly 180 185 190 Gln AsnIle Gly Cys Arg Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys 195 200 205 AspPhe Tyr Ile Cys Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg 210 215 220Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu Pro 225 230235 240 Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu245 250 255 Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe AspTyr 260 265 270 Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val Thr AlaThr Val 275 280 285 Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr Asn Glu ThrArg Gln Leu 290 295 300 Cys Phe Val Val Arg Ser Lys Val Asn Ile Tyr CysSer Asp Asp Gly 305 310 315 320 Ile Trp Ser Glu Trp Ser Asp Lys Gln CysTrp Glu Gly Glu Asp Leu 325 330 335 Ser Lys Lys Thr Leu Leu Arg Phe TrpLeu Pro Phe Gly Phe Ile Leu 340 345 350 Ile Leu Val Ile Phe Val Thr GlyLeu Leu Leu Arg Lys Pro Asn Thr 355 360 365 Tyr Pro Lys Met Ile Pro GluPhe Phe Cys Asp Thr 370 375 380 2 1298 DNA Homo sapiens 2 ggtgcctgtcggcggggaga gaggcaatat caaggtttta aatctcggag aaatggcttt 60 cgtttgcttggctatcggat gcttatatac ctttctgata agcacaacat ttggctgtac 120 ttcatcttcagacaccgaga taaaagttaa ccctcctcag gattttgaga tagtggatcc 180 cggatacttaggttatctct atttgcaatg gcaaccccca ctgtctctgg atcattttaa 240 ggaatgcacagtggaatatg aactaaaata ccgaaacatt ggtagtgaaa catggaagac 300 catcattactaagaatctac attacaaaga tgggtttgat cttaacaagg gcattgaagc 360 gaagatacacacgcttttac catggcaatg cacaaatgga tcagaagttc aaagttcctg 420 ggcagaaactacttattgga tatcaccaca aggaattcca gaaactaaag ttcaggatat 480 ggattgcgtatattacaatt ggcaatattt actctgttct tggaaacctg gcataggtgt 540 acttcttgataccaattaca acttgtttta ctggtatgag ggcttggatc atgcattaca 600 gtgtgttgattacatcaagg ctgatggaca aaatatagga tgcagatttc cctatttgga 660 ggcatcagactataaagatt tctatatttg tgttaatgga tcatcagaga acaagcctat 720 cagatccagttatttcactt ttcagcttca aaatatagtt aaacctttgc cgccagtcta 780 tcttacttttactcgggaga gttcatgtga aattaagctg aaatggagca tacctttggg 840 acctattccagcaaggtgtt ttgattatga aattgagatc agagaagatg atactacctt 900 ggtgactgctacagttgaaa atgaaacata caccttgaaa acaacaaatg aaacccgaca 960 attatgctttgtagtaagaa gcaaagtgaa tatttattgc tcagatgacg gaatttggag 1020 tgagtggagtgataaacaat gctgggaagg tgaagaccta tcgaagaaaa ctttgctacg 1080 tttctggctaccatttggtt tcatcttaat attagttata tttgtaaccg gtctgctttt 1140 gcgtaagccaaacacctacc caaaaatgat tccagaattt ttctgtgata catgaagact 1200 ttccatatcaagagacatgg tattgactca acagtttcca gtcatggcca aatgttcaat 1260 atgagtctcaataaactgaa tttttcttgc gaatgttg 1298 3 30 DNA Artificial Sequencemisc_feature Forward PCR Primer for IL-13Ralpha2 3 aagatttgga agcttatggctttcgtttgc 30 4 30 DNA Artificial Sequence misc_feature Reverse PCRPrimer for IL-13Ralpha2 4 tccctcgaag cttcatgtat cacagaaaaa 30 5 27 DNAArtificial Sequence misc_feature Forward PCR Primer for IL-13Ralpha1 5attattaagc ttatggagtg gccggcg 27 6 27 DNA Artificial Sequencemisc_feature Reverse PCR Primer For IL-13Ralpha1 6 taaccggaag cttcactgagaggcttt 27

What is claimed is:
 1. A method for stimulating a immune responseagainst IL-13Rα2 in a subject having or at risk for developing a diseasehaving cells expressing IL-13Rα2, the method comprising the steps of:(a) formulating an anti-cancer vaccine outside of the subject, thevaccine comprising an agent that can stimulate an immune responseagainst IL-13Rα2 when administered to an animal; and (b) administeringthe vaccine to the subject in an amount sufficient to stimulate animmune response against IL-13Rα2 in the subject.
 2. The method of claim1, wherein the agent that can stimulate an immune response againstIL-13Rox2 comprises a peptide comprising at least seven contiguous aminoacids of SEQ ID NO:1.
 3. The method of claim 1, wherein the agent thatcan stimulate an immune response against IL-13R.α2 is a proteincomprising the amino acid sequence of SEQ ID NO:1.
 4. The method claim1, wherein the vaccine further comprises an adjuvant.
 5. The method ofclaim 4, wherein the adjuvant comprises a substance selected from thegroup consisting of: an aluminum salt; an oil-in-water emulsion; acomposition comprising saponin; a composition comprising a bacterialprotein; and a cytokine.
 6. The method of claim 4, wherein step (b) ofadministering the vaccine to the subject in an amount sufficient tostimulate an immune response against IL-13Rα2 in the subject comprisesadministering the vaccine in at least a first dose and a second dose,wherein said first dose is administered to the subject at least 24 hoursbefore said second dose is administered to the subject.
 7. The method ofclaim 1, wherein the agent that can stimulate an immune response againstIL-13Rα2 comprises a nucleic acid that encodes a peptide comprising atleast seven contiguous amino acids of SEQ ID NO:1.
 8. The method ofclaim 7, wherein the nucleic acid is a naked DNA.
 9. The method of claim7, wherein the nucleic acid is incorporated into an expression vector.10. The method of claim 1, wherein the agent that can stimulate animmune response against IL-13Rα2 comprises a cell expressing a peptidecomprising at least seven contiguous amino acids of SEQ ID NO:1.
 11. Themethod of claim 10, wherein the peptide comprising at least sevencontiguous amino acids of SEQ ID NO:1 is a protein comprising the aminoacid sequence of SEQ ID NO:1.
 12. The method of claim 1, wherein theagent that can stimulate an immune response against IL-13Rα2 comprises acell into which has been introduced a purified nucleic acid that encodesa peptide comprising at least seven contiguous amino acids of SEQ IDNO:1.
 13. The method of claim 1, further comprising the step ofproviding a subject having or at risk for developing a cancer havingcells expressing IL-13Rα2.
 14. The method of claim 13, wherein the cellsexpressing IL-13Rβα2 are glioma cells.
 15. The method of claim 13,wherein the subject is a human being.
 16. A composition for stimulatingan immune response against IL-13Rα2 when administered to an animal, thecomposition comprising: (a) an isolated agent that can stimulate animmune response against IL-13Rα2 when administered to an animal; and (b)a pharmaceutically acceptable carrier.
 17. The composition of claim 16,wherein the agent that can stimulate an immune response against IL-13Rα2when administered to an animal comprises a peptide comprising at leastseven contiguous amino acids of SEQ ID NO:1.
 18. The composition ofclaim 17, wherein the peptide comprising at least seven contiguous aminoacids of SEQ ID NO:1 is a protein comprising the amino acid sequence ofSEQ ID NO:1.
 19. The composition of claim 17, wherein the compositionfurther comprises an adjuvant.
 20. The composition of claim 19, whereinthe adjuvant comprises a substance selected from the group consistingof: an aluminum salt; an oil-in-water emulsion; a composition comprisingsaponin; a composition comprising a bacterial protein; and a cytokine.21. The composition of claim 20, wherein the agent that can stimulate animmune response against IL-13Rα2 when administered to an animalcomprises a nucleic acid that encodes a peptide comprising at leastseven contiguous amino acids of SEQ ID NO:1.
 22. The composition ofclaim 21, wherein the nucleic acid is a naked DNA.
 23. The compositionof claim 21, wherein the nucleic acid is incorporated into an expressionvector.
 24. The composition of claim 20, wherein the agent that canstimulate an immune response against IL-13Rα2 comprises a cellexpressing a peptide comprising at least seven contiguous amino acids ofSEQ ID NO:1.
 25. The method of claim 24, wherein the peptide comprisingat least seven contiguous amino acids of SEQ ID NO:1 is a proteincomprising the amino acid sequence of SEQ ID NO:1.
 26. The compositionof claim 25, wherein the agent that can stimulate an immune responseagainst IL-13Rα2 comprises a cell into which has been introduced apurified nucleic acid that encodes a peptide comprising at least sevencontiguous amino acids of SEQ ID NO:1.
 27. A method for directing anantibody to cells expressing IL-13RU,2 in a subject, the methodcomprising the steps of: (a) formulating a pharmaceutical compositionoutside of a subject, the pharmaceutical composition comprising anantibody that specifically binds IL-13Rα2 and a pharmaceuticallyacceptable carrier; and (b) administering the pharmaceutical compositionto the subject in an amount sufficient to allow the antibody tospecifically bind to the cells expressing IL-13Rα2 in the subject. 28.The method of claim 27, wherein the antibody is a monoclonal antibody.29. The method of claim 27, wherein the antibody is a polyclonalantibody.
 30. A pharmaceutical composition comprising an antibody thatspecifically binds IL-13Rα2 and a pharmaceutically acceptable carrier.31. The pharmaceutical composition of claim 30, wherein the antibody isa monoclonal antibody.
 32. The pharmaceutical composition of claim 30,wherein the antibody is a polyclonal antibody.